Therapeutic uses of inhibitors of RTP801L

ABSTRACT

The present invention provides novel molecules, compositions, methods and uses for treating microvascular disorders, eye diseases respiratory conditions and hearing disorders based upon inhibition of the RTP801L gene and/or protein.

This application is a continuation of U.S. Ser. No. 11/811,112, filedJun. 8, 2007, now U.S. Pat. No. 7,626,015 now allowed, and claims thebenefit of U.S. provisional patent application No. 60/812,229 filed onJun. 9, 2006, both of which are hereby incorporated by reference intheir entirety in this application.

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“172-US1Arevised.ST25.txt,” which is 362 kilobytes in size, and whichwas created Jan. 12, 2010 in the IBM-PCT machine format, having anoperating system compatibility with MS-Windows, which is contained ontwo duplicate compact discs labeled COPY 1 and COPY 2, and also labeledwith the title, names of each inventor, and attorney docket number ofthis application.

FIELD OF THE INVENTION

The present invention relates to novel siRNA molecules which inhibit theRTP801L gene and to the use of such molecules to treat respiratorydisorders of all types (including pulmonary disorders), eye diseases andconditions, microvascular disorders, angiogenesis- and apoptosis-relatedconditions, and hearing impairments.

BACKGROUND OF THE INVENTION

Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD), affects more than 16million Americans and is the fourth highest cause of death in the UnitedStates. Cigarette smoking causes most occurrences of the debilitatingdisease but other environmental factors cannot be excluded (Petty T L.2003. Definition, epidemiology, course, and prognosis of COPD. Clin.Cornerstone, 5-10). Pulmonary emphysema is a major manifestation ofCOPD. Permanent destruction of peripheral air spaces, distal to terminalbronchioles, is the hallmark of emphysema (Tuder R M, et al. Oxidativestress and apoptosis interact and cause emphysema due to vascularendothelial growth factor blocade. Am J Respir Cell Mol Biol, 29:88-97;2003.). Emphysema is also characterized by accumulation of inflammatorycells such as macrophages and neutrophils in bronchioles and alveolarstructures (Petty, 2003).

The pathogenesis of emphysema is complex and multifactorial. In humans,a deficiency of inhibitors of proteases produced by inflammatory cells,such as alpha1-antitrypsin, has been shown to contribute toprotease/antiprotease imbalance, thereby favoring destruction ofalveolar extracellular matrix in cigarette-smoke (CS) induced emphysema(Eriksson, S. 1964. Pulmonary Emphysema and Alpha1-AntitrypsinDeficiency. Acta Med Scand 175:197-205. Joos, L., Pare, P. D., andSandford, A. J. 2002. Genetic risk factors of chronic obstructivepulmonary disease. Swiss Med Wkly 132:27-37). Matrix metalloproteinases(MMPs) play a central role in experimental emphysema, as documented byresistance of macrophage metalloelastase knockout mice against emphysemacaused by chronic inhalation of CS (Hautamaki, et al: Requirement formacrophage elastase for cigarette smoke-induced emphysema in mice.Science 277:2002-2004). Moreover, pulmonary overexpression ofinterleukin-13 in transgenic mice results in MMP- andcathepsin-dependent emphysema (Zheng, T., et al 2000. Inducibletargeting of IL-13 to the adult lung causes matrix metalloproteinase-and Cathepsin-dependent emphysema. J Clin Invest 106:1081-1093). Recentworks describe involvement of septal cell apoptosis in lung tissuedestruction leading to emphysema (Rangasami T, et al. Genetic ablationof Nrf2 enhances susceptibility to cigarette smoke-induced emphysema inmice. Submitted to Journal of Clinincal Investigation.; Tuder R M et al.Oxidative stress and apoptosis interact and cause emphysema due tovascular endothelial growth factor blocade. Am J Respir Cell Mol Biol,29:88-97; 2003.; Yokohori N, Aoshiba K, Nagai A, Increased levels ofcell death and proliferation in alveolar wall cells in patients withpulmonary emphysema. Chest. 2004 February; 125(2):626-32.; Aoshiba K,Yokohori N, Nagai A., Alveolar wall apoptosis causes lung destructionand emphysematous changes. Am J Respir Cell Mol Biol. 2003 May; 28(5):555-62.).

Among the mechanisms that underlie both pathways of lung destruction inemphysema, excessive formation of reactive oxygen species (ROS) shouldbe first of all mentioned. It is well established thatprooxidant/antioxidant imbalance exists in the blood and in the lungtissue of smokers (Hulea S A, et al: Cigarette smoking causesbiochemical changes in blood that are suggestive of oxidative stress: acase-control study. J Environ Pathol Toxicol Oncol. 1995;14(3-4):173-80.; Rahman I, MacNee W. Lung glutathione and oxidativestress: implications in cigarette smoke-induced airway disease. Am JPhysiol. 1999 December; 277(6 Pt 1):L1067-88.; MacNee W.Oxidants/antioxidants and COPD. Chest. 2000 May; 117(5 Suppl1):303S-17S.; Marwick J A, Kirkham P, Gilmour P S, Donaldson K, MacNEEW, Rahman I. Cigarette smoke-induced oxidative stress and TGF-beta1increase p21waf1/cip1 expression in alveolar epithelial cells. Ann NYAcad Sci. 2002 November; 973:278-83.; Aoshiba K, Koinuma M, Yokohori N,Nagai A. Immunohistochemical evaluation of oxidative stress in murinelungs after cigarette smoke exposure. Inhal Toxicol. 2003 September;15(10):1029-38.; Dekhuijzen P N. Antioxidant properties ofN-acetylcysteine: their relevance in relation to chronic obstructivepulmonary disease. Eur Respir J. 2004 April; 23(4):629-36.; Tuder R M,Zhen L, Cho C Y, Taraseviciene-Stewart L, Kasahara Y, Salvemini D,Voelkel N F, and Flores S C. Oxidative stress and apoptosis interact andcause emphysema due to vascular endothelial growth factor blocade. Am JRespir Cell Mol Biol, 29:88-97; 2003.). After one hour exposure of miceto CS, there is a dramatic increase of 8-hydroxy-2′-deoxyguanosine(8-OHdG) in the alveolar epithelial cells, particularly of type II (seeInhal Toxicol. 2003 September; 15(10):1029-38. above).

Overproduced reactive oxygen species are known for their cytotoxicactivity, which stems from a direct DNA damaging effect and from theactivation of apoptotic signal transduction pathways (Takahashi A,Masuda A, Sun M, Centonze V E, Herman B. Oxidative stress-inducedapoptosis is associated with alterations in mitochondrial caspaseactivity and Bcl-2-dependent alterations in mitochondrial pH (pHm).Brain Res Bull. 2004 Feb. 15; 62(6):497-504.; Taniyama Y, Griendling KK. Reactive oxygen species in the vasculature: molecular and cellularmechanisms. Hypertension. 2003 December; 42(6):1075-81. Epub 2003 Oct.27.; Higuchi Y. Chromosomal DNA fragmentation in apoptosis and necrosisinduced by oxidative stress. Biochem Pharmacol. 2003 Oct. 15;66(8):1527-35.; Punj V, Chakrabarty A M. Redox proteins in mammaliancell death: an evolutionarily conserved function in mitochondria andprokaryotes. Cell Microbiol. 2003 April; 5(4):225-31.; Ueda S, MasutaniH, Nakamura H, Tanaka T, Ueno M, Yodoi J. Redox control of cell death.Antioxid Redox Signal. 2002 June; 4(3):405-14.).

ROS's are not only cytotoxic per se but are also proinflammatorystimuli, being prominent activators of redox-sensitive transcriptionfactors NFkB and AP-1 (reviewed in Rahman I. Oxidative stress and genetranscription in asthma and chronic obstructive pulmonary disease:antioxidant therapeutic targets. Curr Drug Targets Inflamm Allergy. 2002September; 1(3):291-315.). Both transcription factors are, in turn,strongly implicated in stimulation of transcription of proinflammatorycytokines (reviewed in Renard P, Raes M. The proinflammatorytranscription factor NFkappaB: a potential target for noveltherapeutical strategies. Cell Biol Toxicol. 1999; 15(6):341-4.; LentschA B, Ward P A. The NFkappaBb/IkappaB system in acute inflammation. ArchImmunol Ther Exp (Warsz). 2000; 48(2):59-63) and matrix degradingproteinases (Andela V B, Gordon A H, Zotalis G, Rosier R N, Goater J J,Lewis G D, Schwarz E M, Puzas J E, O'Keefe R J. NFkappaB: a pivotaltranscription factor in prostate cancer metastasis to bone. Clin Orthop.2003 October; (415 Suppl):S75-85.; Fleenor D L, Pang I H, Clark A F.Involvement of AP-1 in interleukin-1alpha-stimulated MMP-3 expression inhuman trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2003 August;44(8):3494-501.; Ruhul Amin A R, Senga T, Oo M L, Thant A A, HamaguchiM. Secretion of matrix Metalloproteinase-9 by the proinflammatorycytokine, IL-1beta: a role for the dual signalling pathways, Akt andErk. Genes Cells. 2003 June; 8(6):515-23.). Proinflammatory cytokines,in turn, serve as attractors of inflammatory cells that also secretematrix degrading enzymes, cytokines and reactive oxygen species. Thus,it appears that a pathogenic factor, like e.g. CS, triggers apathological network where reactive oxygen species act as majormediators of lung destruction. Both reactive oxygen species (ROS) frominhaled cigarette smoke and those endogenously formed by inflammatorycells contribute to an increased intrapulmonary oxidant burden.

One additional pathogenic factor with regards to COPD pathogenesis isthe observed decreased expression of VEGF and VEGFRII in lungs ofemphysematous patients (Yasunori Kasahara, Rubin M. Tuder, Carlyne D.Cool, David A. Lynch, Sonia C. Flores, and Norbert F. Voelkel.Endothelial Cell Death and Decreased Expression of Vascular EndothelialGrowth Factor and Vascular Endothelial Growth Factor Receptor 2 inEmphysema. Am J Respir Crit Care Med Vol 163. pp 737-744, 2001).Moreover, inhibition of VEGF signaling using chemical VEGFR inhibitorleads to alveolar septal endothelial and then to epithelial cellapoptosis, probably due to disruption of intimate structural/functionalconnection of both types of cells within alveoli (Yasunori Kasahara,Rubin M. Tuder, Laimute Taraseviciene-Stewart, Timothy D. Le Cras,Steven Abman, Peter K. Hirth, Johannes Waltenberger, and Norbert F.Voelkel. Inhibition of VEGF receptors causes lung cell apoptosis andemphysema. J. Clin. Invest. 106:1311-1319 (2000).; Voelkel N F, Cool CD. Pulmonary vascular involvement in chronic obstructive pulmonarydisease. Eur Respir J Suppl. 2003 November; 46:28s-32s).

Macular Degeneration

The most common cause of decreased best-corrected vision in individualsover 65 years of age in the US is the retinal disorder known asage-related macular degeneration (AMD). As AMD progresses, the diseaseis characterized by loss of sharp, central vision. The area of the eyeaffected by AMD is the Macula—a small area in the center of the retina,composed primarily of photoreceptor cells. So-called “dry” AMD,accounting for about 85%-90% of AMD patients, involves alterations ineye pigment distribution, loss of photoreceptors and diminished retinalfunction due to overall atrophy of cells. So-called “wet” AMD involvesproliferation of abnormal choroidal vessels leading to clots or scars inthe sub-retinal space. Thus, the onset of wet AMD occurs because of theformation of an abnormal choroidal neovascular network (choroidalneovascularization, CNV) beneath the neural retina. The newly formedblood vessels are excessively leaky. This leads to accumulation ofsubretinal fluid and blood leading to loss of visual acuity. Eventually,there is total loss of functional retina in the involved region, as alarge disciform scar involving choroids and retina forms. While dry AMDpatients may retain vision of decreased quality, wet AMD often resultsin blindness. (Hamdi & Kenney, Age-related Macular degeneration—a newviewpoint, Frontiers in Bioscience, e305-314, May 2003). CNV occurs notonly in wet AMD but also in other ocular pathologies such as ocularhistoplasmosis syndrome, angiod streaks, ruptures in Bruch's membrane,myopic degeneration, ocular tumors and some retinal degenerativediseases.

Various studies conducted have determined several risk factors for AMD,such as smoking, aging, family history (Milton, Am J Ophthalmol 88, 269(1979); Mitchell et al., Ophthalmology 102, 1450-1460 (1995); Smith etal., Ophthalmology 108, 697-704 (2001)) sex (7-fold higher likelihood infemales: Klein et al., Ophthalmology 99, 933-943 (1992) and race (whitesare most susceptible). Additional risk factors may include eyecharacteristics such as farsightedness (hyperopia) and light-coloredeyes, as well as cardiovascular disease and hypertension. Evidence ofgenetic involvement in the onset progression of the disease has alsobeen documented (see Hamdi & Kenney above).

Two companies, Acuity Pharmaceuticals and Sirna Therapeutics, have bothrecently filed an IND for siRNA molecules inhibiting VEGF and VEGF-R1(Flt-1), respectively, for treatment of AMD. These molecules are termedCand5 inhibitor and 027 inhibitor respectively.

Glaucoma

Glaucoma is one of the leading causes of blindness in the world. Itaffects approximately 66.8 million people worldwide. At least 12,000Americans are blinded by this disease each year (Kahn and Milton, Am JEpidemiol. 1980 111(6):769-76). Glaucoma is characterized by thedegeneration of axons in the optic nerve head, primarily due to elevatedintraocular pressure (IOP). One of the most common forms of glaucoma,known as primary open-angle glaucoma (POAG), results from the increasedresistance of aqueous humor outflow in the trabecular meshwork (TM),causing IOP elevation and eventual optic nerve damage.

Microvascular Disorders

Microvascular disorders are composed of a broad group of conditions thatprimarily affect the microscopic capillaries and lymphatics and aretherefore outside the scope of direct surgical intervention.Microvascular disease can be broadly grouped into the vasospastic, thevasculitis and lymphatic occlusive. Additionally, many of the knownvascular conditions have a microvascular element to them.

-   -   Vasospastic Disease—Vasospastic diseases are a group of        relatively common conditions where, for unknown reasons, the        peripheral vasoconstrictive reflexes are hypersensitive. This        results in inappropriate vasoconstriction and tissue ischaemia,        even to the point of tissue loss. Vasospastic symptoms are        usually related to temperature or the use of vibrating machinery        but may be secondary to other conditions.    -   Vasculitic Disease—Vasculitic diseases are those that involve a        primary inflammatory process in the microcirculation. Vasculitis        is usually a component of an autoimmune or connective tissue        disorder and is not generally amenable to surgical treatment but        requires immunosuppressive treatment if the symptoms are severe.    -   Lymphatic Occlusive Disease—Chronic swelling of the lower or        upper limb (lymphoedema) is the result of peripheral lymphatic        occlusion. This is a relatively rare condition that has a large        number of causes, some inherited, some acquired. The mainstays        of treatment are correctly fitted compression garments and the        use of intermittent compression devices.

Microvascular Pathologies Associated with Diabetes

Diabetes is the leading cause of blindness, the number one cause ofamputations and impotence, and one of the most frequently occurringchronic childhood diseases. Diabetes is also the leading cause ofend-stage renal disease in the United States, with a prevalence rate of31% compared with other renal diseases. Diabetes is also the mostfrequent indication for kidney transplantation, accounting for 22% ofall transplantation operations.

In general, diabetic complications can be classified broadly asmicrovascular or macrovascular disease. Microvascular complicationsinclude neuropathy (nerve damage), nephropathy (kidney disease) andvision disorders (eg retinopathy, glaucoma, cataract and cornealdisease). In the retina, glomerulus, and vasa nervorum, similarpathophysiologic features characterize diabetes-specific microvasculardisease.

Microvascular pathologies associated with diabetes are defined as adisease of the smallest blood vessels (capillaries) that may occur e.g.in people who have had diabetes for a long time. The walls of thevessels become abnormally thick but weak. They, therefore, bleed, leakprotein and slow the flow of blood through the body.

Clinical and animal model data indicate that chronic hyperglycemia isthe central initiating factor for all types of diabetic microvasculardisease. Duration and magnitude of hyperglycemia are both stronglycorrelated with the extent and rate of progression of diabeticmicrovascular disease. Although all diabetic cells are exposed toelevated levels of plasma glucose, hyperglycemic damage is limited tothose cell types (e.g., endothelial cells) that develop intracellularhyperglycemia. Endothelial cells develop intracellular hyperglycemiabecause, unlike many other cells, they cannot down-regulate glucosetransport when exposed to extracellular hyperglycemia. Thatintracellular hyperglycemia is necessary and sufficient for thedevelopment of diabetic pathology is further demonstrated by the factthat overexpression of the GLUT1 glucose transporter in mesangial cellscultured in a normal glucose milieu mimics the diabetic phenotype,inducing the same increases in collagen type IV, collagen type I, andfibronectin gene expression as diabetic hyperglycemia.

Abnormal Endothelial Cell Function: Early in the course of diabetesmellitus, before structural changes are evident, hyperglycemia causesabnormalities in blood flow and vascular permeability in the retina,glomerulus, and peripheral nerve vasa nervorum. The increase in bloodflow and intracapillary pressure is thought to reflecthyperglycemia-induced decreased nitric oxide (NO) production on theefferent side of capillary beds, and possibly an increased sensitivityto angiotensin II. As a consequence of increased intracapillary pressureand endothelial cell dysfunction, retinal capillaries exhibit increasedleakage of fluorescein and glomerular capillaries have an elevatedalbumin excretion rate (AER). Comparable changes occur in the vasavasorum of peripheral nerve. Early in the course of diabetes, increasedpermeability is reversible; as time progresses, however, it becomesirreversible.

Increased Vessel Wall Protein Accumulation

The common pathophysiologic feature of diabetic microvascular disease isprogressive narrowing and eventual occlusion of vascular lumina, whichresults in inadequate perfusion and function of the affected tissues.Early hyperglycemia-induced microvascular hypertension and increasedvascular permeability contribute to irreversible microvessel occlusionby three processes:

-   -   The first is an abnormal leakage of periodic acid-Schiff        (PAS)-positive, carbohydrate-containing plasma proteins, which        are deposited in the capillary wall and which may stimulate        perivascular cells such as pericytes and mesangial cells to        elaborate growth factors and extracellular matrix.    -   The second is extravasation of growth factors, such as        transforming growth factor β1 (TGF-β1), which directly        stimulates overproduction of extracellular matrix components,        and may induce apoptosis in certain complication-relevant cell        types.    -   The third is hypertension-induced stimulation of pathologic gene        expression by endothelial cells and supporting cells, which        include glut-1 glucose transporters, growth factors, growth        factor receptors, extracellular matrix components, and adhesion        molecules that can activate circulating leukocytes. The        observation that unilateral reduction in the severity of        diabetic microvascular disease occurs on the side with        ophthalmic or renal artery stenosis is consistent with this        concept.

Microvascular Cell Loss and Vessel Occlusion

The progressive narrowing and occlusion of diabetic microvascular luminaare also accompanied by microvascular cell loss. In the retina, diabetesmellitus induces programmed cell death of Muller cells and ganglioncells, pericytes, and endothelial cells. In the glomerulus, decliningrenal function is associated with widespread capillary occlusion andpodocyte loss, but the mechanisms underlying glomerular cell loss arenot yet known. In the vasa nervorum, endothelial cell and pericytedegeneration occur, and these microvascular changes appear to precedethe development of diabetic peripheral neuropathy. The multifocaldistribution of axonal degeneration in diabetes supports a causal rolefor microvascular occlusion, but hyperglycemia-induced decreases inneurotrophins may contribute by preventing normal axonal repair andregeneration.

Another common feature of diabetic microvascular disease has been termedhyperglycemic memory, or the persistence or progression ofhyperglycemia-induced microvascular alterations during subsequentperiods of normal glucose homeostasis. The most striking example of thisphenomenon is the development of severe retinopathy in histologicallynormal eyes of diabetic dogs that occurred entirely during a 2.5-yearperiod of normalized blood glucose that followed 2.5 years ofhyperglycemia. Hyperglycemia-induced increases in selected matrix genetranscription also persist for weeks after restoration of normoglycemiain vivo, and a less pronounced, but qualitatively similar, prolongationof hyperglycemia-induced increase in selected matrix gene transcriptionoccurs in cultured endothelial cells.

For further information, see “Shared pathophysiologic features ofmicrovascular complications of diabetes” (Larsen: Williams Textbook ofEndocrinology, 10th ed., Copyright ©2003 Elsevier).

Microvascular complications occur not only in overt diabetes but arealso due to Impaired Glucose Tolerance (IGT). Microvascularcomplications of IGT: neuropathy, retinopathy, and renalmicroproteinuria.

Diabetic Neuropathy

Diabetic neuropathies are neuropathic disorders (peripheral nervedamage) that are associated with diabetes mellitus. These conditionsusually result from diabetic microvascular injury involving small bloodvessels that supply nerves (vasa nervorum). Relatively common conditionswhich may be associated with diabetic neuropathy include third nervepalsy; mononeuropathy; mononeuropathy multiplex; diabetic amyotrophy; apainful polyneuropathy; autonomic neuropathy; and thoracoabdominalneuropathy and the most common form, peripheral neuropathy, which mainlyaffects the feet and legs. There are four factors involved in thedevelopment of diabetic neuropathy: microvascular disease, advancedglycated end products, protein kinase C, and the polyol pathway.

Microvascular Disease in Diabetic Neuropathy

Vascular and neural diseases are closely related and intertwined. Bloodvessels depend on normal nerve function, and nerves depends on adequateblood flow. The first pathological change in the microvasculature isvasoconstriction. As the disease progresses, neuronal dysfunctioncorrelates closely with the development of vascular abnormalities, suchas capillary basement membrane thickening and endothelial hyperplasia,which contribute to diminished oxygen tension and hypoxia. Neuronalischemia is a well-established characteristic of diabetic neuropathy.Vasodilator agents (e.g., angiotensin-converting-enzyme inhibitors,alphal-antagonists) can lead to substantial improvements in neuronalblood flow, with corresponding improvements in nerve conductionvelocities. Thus, microvascular dysfunction occurs early in diabetes,parallels the progression of neural dysfunction, and may be sufficientto support the severity of structural, functional, and clinical changesobserved in diabetic neuropathy. Peripheral neuropathy (legs),sensorimotor neuropathy is a significant component in the pathogenesisof leg ulcers in diabetes.

Neuropathy is a common complication of diabetes occurring over time inmore than half of patients with type 2 diabetes. Nerve conductionstudies demonstrate that neuropathy is already present in 10-18% ofpatients at the time of diabetes diagnosis, suggesting that peripheralnerve injury occurs at early stages of disease and with milder glycemicdysregulation. The concept that neuropathy is an early clinical sign ofdiabetes was proposed >40 years ago, and most studies report anassociation between IGT and neuropathy. Most patients with IGT andassociated neuropathy have a symmetric, distal sensory polyneuropathywith prominent neuropathic pain. IGT neuropathy (Microvascularcomplications of impaired glucose tolerance—Perspectives in Diabetes, J.Robinson Singleton, in Diabetes Dec. 1, 2003) is phenotypically similarto early diabetic neuropathy, which also causes sensory symptoms,including pain, and autonomic dysfunction. In a survey of 669 patientswith early diabetic neuropathy, sensory symptoms were present in >60%,impotence in nearly 40%, and other autonomic involvement in 33%, butevidence of motor involvement in only 12%. These clinical findingssuggest prominent early involvement of the small unmyelinated nervefibers that carry pain, temperature, and autonomic signals. Directquantitation of unmyelinated intraepidermal nerve fibers from skinbiopsies shows similar fiber loss and altered morphology in patientswith neuropathy associated with IGT and early diabetes.

Autonomic dysfunction, particularly erectile dysfunction and alteredcardiac vagal response, are common early features of neuropathic injuryin diabetes. Work with IGT patients also suggests prevalent vagaldysautonoinia: separate studies have found abnormal heart rate recoveryfollowing exercise, blunted R-R interval variability to deep breathing,and reduced expiration to inspiration ratio (all measures of vagaldysautonomia) in a greater fraction of IGT patients than age-matchednormoglycemic control subjects.

Nerve damage in diabetes affects the motor, sensory, and autonomicfibers. Motor neuropathy causes muscle weakness, atrophy, and paresis.Sensory neuropathy leads to loss of the protective sensations of pain,pressure, and heat. The absence of pain leads to many problems in theinsensate foot, including ulceration, unperceived trauma, and Charcotneuroarthropathy. The patient may not seek treatment until after thewound has advanced. A combination of sensory and motor dysfunction cancause the patient to place abnormal stresses on the foot, resulting intrauma, which may lead to infection.

Autonomic sympathetic neuropathy causes vasodilation and decreasedsweating, which results in warm, overly dry feet that are particularlyprone to skin breakdown, as well as functional alterations inmicrovascular flow. Autonomic dysfunction (and denervation of dermalstructures) also results in loss of skin integrity, which provides anideal site for microbial invasion. The neuropathic foot does notulcerate spontaneously; rather, it is the combination of some form oftrauma accompanied by neuropathy.

Microvascular dysfunction occurs early in diabetes, parallels theprogression of neural dysfunction, and may be sufficient to support theseverity of structural, functional, and clinical changes observed indiabetic neuropathy.

Advanced glycated end products—Elevated intracellular levels of glucosecause a non-enzymatic covalent bonding with proteins, which alters theirstructure and destroys their function. Certain of these glycatedproteins are implicated in the pathology of diabetic neuropathy andother long term complications of diabetes.

Protein kinase C (PKC)—PKC is implicated in the pathology of diabeticneuropathy. Increased levels of glucose cause an increase inintracellular diacylglycerol, which activates PKC. PKC inhibitors inanimal models will increase nerve conduction velocity by increasingneuronal blood flow.

Sensorimotor Polyneuropathy

Longer nerve fibers are affected to a greater degree than shorter ones,because nerve conduction velocity is slowed in proportion to a nerve'slength. In this syndrome, decreased sensation and loss of reflexesoccurs first in the toes bilaterally, then extends upward. It is usuallydescribed as glove-stocking distribution of numbness, sensory loss,dysesthesia and nighttime pain. The pain can feel like burning, prickingsensation, achy or dull. Pins and needles sensation is common. Loss ofproprioception, that is, the sense of where a limb is in space, isaffected early. These patients cannot feel when they are stepping on aforeign body, like a splinter, or when they are developing a callousfrom an ill-fitting shoe. Consequently, they are at risk for developingulcers and infections on the feet and legs, which can lead toamputation. Similarly, these patients can get multiple fractures of theknee, ankle or foot, and develop a Charcot joint. Loss of motor functionresults on dorsiflexion contractures of the toes, so called hammertoes.These contractures occur not only in the foot but also in the hand.

Autonomic Neuropathy

The autonomic nervous system is composed of nerves serving the heart, GItract and urinary system. Autonomic neuropathy can affect any of theseorgan systems. The most commonly recognized autonomic dysfuction indiabetics is orthostatic hypotension, or the uncomfortable sensation offainting when a patient stands up. In the case of diabetic autonomicneuropathy, it is due to the failure of the heart and arteries toappropriately adjust heart rate and vascular tone to keep bloodcontinually and fully flowing to the brain. This symptom is usuallyaccompanied by a loss of sinus respiratory variation, that is, the usualchange in heart rate seen with normal breathing. When these two findingsare present, cardiac autonomic neuropathy is present.

GI tract manifestations include delayed gastric emptying, gastroparesis,nausea, bloating, and diarrhea. Because many diabetics take oralmedication for their diabetes, absorption of these medicines is greatlyaffected by the delayed gastric emptying. This can lead to hypoglycemiawhen an oral diabetic agent is taken before a meal and does not getabsorbed until hours, or sometimes days later, when there is normal orlow blood sugar already. Sluggish movement of the small instestine cancause bacterial overgrowth, made worse by the presence of hyperglycemia.This leads to bloating, gas and diarrhea.

Urinary symptoms include urinary frequency, urgency, incontinence andretention. Again, because of the retention of sweet urine, urinary tractinfections are frequent. Urinary retention can lead to bladderdiverticula, stones, reflux nephropathy.

Cranial Neuropathy

When cranial nerves are affected, oculomotor (3rd) neuropathies are mostcommon. The oculomotor nerve controls all of the muscles that move theeye with the exception of the lateral rectus and superior obliquemuscles. It also serves to constrict the pupil and open the eyelid. Theonset of a diabetic third nerve palsy is usually abrupt, beginning withfrontal or periorbital pain and then diplopia. All of the oculomotormuscles innervated by the third nerve may be affected, except for thosethat control pupil size. The sixth nerve, the abducens nerve, whichinnervates the lateral rectus muscle of the eye (moves the eyelaterally), is also commonly affected but fourth nerve, the trochlearnerve, (innervates the superior oblique muscle, which moves the eyedownward) involvement is unusual. Mononeuropathies of the thoracic orlumbar spinal nerves can occur and lead to painful syndromes that mimicmyocardial infarction, cholecystitis or appendicitis. Diabetics have ahigher incidence of entrapment neuropathies, such as carpal tunnelsyndrome.

Diabetic Limb Ischemia and Diabetic Foot Ulcers

Diabetes and pressure can impair microvascular circulation and lead tochanges in the skin on the lower extremities, which in turn, can lead toformation of ulcers and subsequent infection. Microvascular changes leadto limb muscle microangiopathy, as well as a predisposition to developperipheral ischemia and a reduced angiogenesis compensatory response toischemic events. Microvascular pathology exacerbates Peripheral VascularDisease (PVD) (or Peripheral Arterial Disease (PAD) or Lower ExtremityArterial Disease (LEAD)—a MACROvascular complication—narrowing of thearteries in the legs due to atherosclerosis. PVD occurs earlier indiabetics, is more severe and widespread, and often involvesintercurrent microcirculatory problems affecting the legs, eyes, andkidneys.

Foot ulcers and gangrene are frequent comorbid conditions of PAD.Concurrent peripheral neuropathy with impaired sensation make the footsusceptible to trauma, ulceration, and infection. The progression of PADin diabetes is compounded by such comorbidity as peripheral neuropathyand insensitivity of the feet and lower extremities to pain and trauma.With impaired circulation and impaired sensation, ulceration andinfection occur. Progression to osteomyelitis and gangrene maynecessitate amputation.

Persons with diabetes are up to 25 times more likely than nondiabeticpersons to sustain a lower limb amputation, underscoring the need toprevent foot ulcers and subsequent limb loss.

Diabetic foot ulcers may occur not only in conjunction with PAD but mayalso be associated with neuropathy, venous insufficiency (varicoseveins), trauma, and infection. PAD contributes to these other conditionsin producing or precipitating foot ulcers. Foot ulcers do notnecessarily represent progression of PAD, as they may occur in thepresence of adequate clinical peripheral arterial perfusion.Patient-based studies indicate an increased risk of foot ulceration indiabetic patients who have peripheral neuropathy and a high plantar footpressure. The prevalence of a history of ulcers or sores on the foot orankles was 15% of all diabetic patients in the population-based study insouthern Wisconsin. The prevalence was higher for diabetic individualsdiagnosed at age <30 years, was slightly higher in men (16%) than inwomen (13%), and was greater in insulin-treated diabetic patients (17%)than in patients not taking insulin (10%). The prevalence increased withage, especially in diabetic patients diagnosed at age <30 years. Inpatient studies from Europe, prevalence of foot ulcers in diabeticpatients was 3% in those age <50 years, 7% in those age <60 years, and14% in those age <80 years. Prevalence was greater in males than infemales at age 70 years.

In diabetic patients, foot ischemia and infection are serious and evenlife-threatening occurrences; however, neuropathy is the most difficultcondition to treat. The medical and surgical literature concerning allaspects of the clinical and pathological manifestations of the diabeticfoot is overwhelming. Neuropathy, angiopathy, retinopathy, andnephropathy, alone or in combination and in varying degrees of severity,may influence the treatment of the diabetic foot.

Every year, 82,000 limb amputations are performed in patients withdiabetes mellitus. The majority of these amputations are performed inthe elderly population. Amputations resulting from diabetes may arisefrom multiple etiologies, including foot ulcers, ischemia, venous legulcers (ie, those secondary to venous reflux), and heel ulcers (ie,those resulting from untreated pressure ulcers in the heel). Themajority of these amputations originate from ulcers. The prevalence offoot ulcers among patients with diabetes is 12%. In addition, the20-year cumulative incidence of lower-extremity ulcers in patients withtype 1 diabetes is 9.9%. Diabetes-induced limb amputations result in a5-year mortality rate of 39% to 68% and are associated with an increasedrisk of additional amputations. The length of hospital stay isapproximately 60% longer among patients with diabetic foot ulcers, ascompared with those without ulcers.

Diabetic neuropathy impairs the nerve axon reflex that depends onhealthy C-fiber nociceptor function and causes local vasodilation inresponse to a painful stimulus. This condition further compromises thevasodilatory response present in conditions of stress, such as injury orinflammation, in the diabetic neuropathic foot. This impairment maypartially explain why some ulcers in the diabetic neuropathic foot areeither slow to heal or fail to heal at all, despite successfullower-extremity revascularization.

The most common causal pathway to diabetic foot ulceration can thus beidentified as the combination of neuropathy (sensory loss), deformity(eg, prominent metatarsal heads), and trauma (eg, ill-fitting footwear).

Most surgeons prefer to perform popliteal or tibial arterial bypassbecause of inferior rates of limb salvage and patency compared with moreproximal procedures. If popliteal or tibial arterial bypass is unable torestore a palpable foot pulse, pedal bypass has been reported to providea more durable and effective limb-salvage procedure for patients withdiabetes and ischemic foot wounds]. Even extensive multisegmentocclusive disease in patients with diabetes does not present animpediment to foot salvage. Whereas serious wound complications may havedisastrous results, they are uncommon after pedal bypass grafting.Adequate control of preexisting foot infection and careful grafttunneling have been shown to be effective in avoiding furthercomplications. Angioplasty in the lower extremity is becoming moreprogressively utilized. However, it must be emphasized that forangioplasty to be effective, a distal vessel or feeding vessel must bepatent if the more proximal angioplasty is to succeed.

While diabetic ulcers/limb pathologies may be managed in some patients(by Debridement, antibiotic treatment, use of preparations to stimulategranulation tissue (new collagen and angiogenesis) and reduction ofbacterial burden in the wound), it would be beneficial to have apharmaceutical composition that could better treat these conditionsand/or alleviate the symptoms.

For further information, see American Journal of Surgery, Volume 187 •Number 5 Suppl 1 • May 1, 2004, Copyright ©2004 Elsevier.

Coronary Microvascular Dysfunction in Diabetes

The correlation between histopathology and microcirculatory dysfunctionin diabetes is well known from old experimental studies and fromautopsy, where thickening of the basal membrane, perivascular fibrosis,vascular rarefication, and capillary hemorrhage are frequently found. Itremains difficult to confirm these data in vivo, although a recent paperdemonstrated a correlation between pathology and ocular micorovasculardysfunction (Am J Physiol 2003; 285). A large amount of clinicalstudies, however, indicate that not only overt diabetes but alsoimpaired metabolic control may affect coronary microcirculation (HypertRes 2002; 25:893). Werner alluded to the important paper by Sambuceti etal (Circulation 2001; 104:1129) showing the persistence of microvasculardysfunction in patients after successful reopening of the infarctrelated artery, and which may explain the increased cardiovascularmorbidity and mortality in these patients. There is mounting evidencefrom large acute reperfusion studies that morbidity and mortality areunrelated to the reopening itself of the infarct related artery, butmuch more dependent on the TIMI flow+/− myocardial blush (Stone 2002;Feldmann Circulation 2003). Herrmann indicated, among others, that theintegrity of the coronary microcirculation is probably the mostimportant clincal and prognostic factor in this context (Circulation2001). The neutral effect of protection devices (no relevant change forTIMI flow, for ST resolution, or for MACE) may indicate that afunctional impairment of microcirculation is the major determinant ofprognosis. There is also increasing evidence that coronary microvasculardysfunction plays a major role in non obstructive CAD. Coronaryendothelial dysfunction remains a strong prognostic predictor in thesepatients.

Diabetic Nephropathy (Renal Dysfunction in Patients with Diabetes)

Diabetic nephropathy encompasses microalbuminuria (a microvasculardisease effect), proteinuria and ESRD. Diabetes is the most common causeof kidney failure, accounting for more than 40 percent of new cases.Even when drugs and diet are able to control diabetes, the disease canlead to nephropathy and kidney failure. Most people with diabetes do notdevelop nephropathy that is severe enough to cause kidney failure. About16 million people in the United States have diabetes, and about 100,000people have kidney failure as a result of diabetes.

Diabetic Retinopathy

In the diabetic state, hyperglycemia leads to decreased retinal bloodflow, retinal hyperpermeability, delays in photoreceptor nerveconduction, and retinal neuronal cell death. In short duration diabetes,neuronal cell death has been identified within the inner nuclear layerof the retina. Specifically, apoptosis has been localized to glial cellssuch as Mueller cells and astrocytes and has been shown to occur within1 month of diabetes in the STZ-induced diabetic rat model. The cause ofthese events is multi-factorial including activation of thediacylglycerobPKC pathway, oxidative stress, and nonenzymaticglycosylation. The combination of these events renders the retinahypoxic and ultimately leads to the development of diabetic retinopathy.One possible connection between retinal ischemia and the early changesin the diabetic retina is the hypoxia-induced production of growthfactors such as VEGF. The master regulator of the hypoxic response hasbeen identified as hypoxia inducible factor-1 (HIF-1), which controlsgenes that regulate cellular proliferation and angiogenesis. Priorstudies have demonstrated that inhibition of HIF-1 ubiquitination leadsto binding with hypoxia responsive elements (HRE) and production of VEGFmRNA.

Diabetic Retinopathy is defined as the progressive dysfunction of theretinal vasculature caused by chronic hyperglycemia. Key features ofdiabetic retinopathy include microaneurysms, retinal hemorrhages,retinal lipid exudates, cotton-wool spots, capillary nonperfusion,macular edema and neovascularization. Associated features includevitreous hemorrhage, retinal detachment, neovascular glaucoma, prematurecataract and cranial nerve palsies.

There are 16 million people in the US with Type 1 and Type 2 diabetes.Within 15 years, 80% of Type 1 patients have developed diabeticretinopathy while 84% of Type 2 diabetic patients develop retinopathywithin 19 years. These numbers constitute a significant market fortherapeutic agents aimed at ocular diseases of neovasculature. Thedevelopment of diabetic retinopathy is time-dependent. Despite optimalblood sugar control, patients with long-standing disease can be expectedto eventually develop some form of retinopathy. The National Society toPrevent Blindness has estimated that 4 to 6 million diabetics in theU.S. have diabetic retinopathy. The estimated annual incidence of newcases of proliferative diabetic retinopathy and diabetic macular edemaare 65,000 and 75,000, respectively, with a prevalence of 700,000 and500,000 respectively. Diabetic retinopathy causes from 12,000 to 24,000new cases of blindness in the US every year. Retinopathy is treated bysurgical methods, effective in reducing severe vision loss, but thelasered portions of the retina are irreversibly destroyed. There are nodrug treatments available.

A microvascular disease that primarily affects the capillaries, diabetesmellitus affects the eye by destroying the vasculature in theconjunctiva, retina and central nervous system. Patients may presentwith histories of long-standing injected bulbar conjunctivae along withsystemic complaints of weight loss despite larger than normal appetite(polyphasia), abnormal thirst (polydypsia) and abnormally frequenturination (polyuria).

Fluctuating visual acuity secondary to unstable blood sugar is a commonocular sign. Swelling within the crystalline lens results in largesudden shifts in refraction as well as premature cataract formation.Changes in visual acuity will depend upon the severity and stage of thedisease.

In the retina, weakening of the arterioles and capillaries may result inthe characteristic appearance of intraretinal dot and blot hemorrhages,exudates, intraretinal microvascular abnormalities (IRMA)microaneurysms, edema and cotton wool infarcts. Proliferative diabeticretinopathy is the result of severe vascular compromise and is visibleas neovascularization of the disc (NVD), neovascularization elsewhere(NVE) and neovascularization of the iris (NVI, or rubeosis irides).Neurological complications include palsies of the third, fourth andsixth cranial nerves as well as diabetic papillitis and facial nerveparalysis.

Diabetes mellitus is a genetically influenced group of diseases thatshare glucose intolerance. It is characterized as a disorder ofmetabolic regulation as a result of deficient or malfunctioning insulinor deficient or malfunctioning cellular insulin receptors.

Biochemistry involving the formation of sorbitol plays a role in thedestruction of pericytes, which are cells that support the vascularendothelium. As the supportive pericytes perish, capillary endotheliumbecomes compromised, resulting in the vascular leakage of blood, proteinand lipid. This, in combination with thickened, glucose-laden blood,produces vascular insufficiency, capillary nonperfusion, retinalhypoxia, altered structure and decreased function. The formation andrelease of vasoproliferative factors which play a role in the genesis ofretinal neovascularization are poorly understood.

Most non-vision threatening sequelae of diabetes resolve spontaneouslyover the course of weeks to months following medical control. In caseswhere there are large refractive changes, patients may require atemporary spectacle prescription until the refraction stabilizes. Whenretinopathy threatens the macula or when new blood vessels proliferate,the patient may be referred for laser photocoagulation. The DiabeticRetinopathy Study (DRS) has conclusively proven that panretinalphotocoagulation was successful in reducing the risk of severe visionloss in high-risk patients. It defined the high-risk characteristics as:(1) Neovascularization of the optic disc (NVD) one-quarter to one-thirdof a disc diameter in size and (2) Neovascularization elsewhere (NVE)with any vitreous hemorrhage.

Diabetic Macular Edema (DME)

DME is a complication of diabetic retinopathy, a disease affecting theblood vessels of the retina. Diabetic retinopathy results in multipleabnormalities in the retina, including retinal thickening and edema,hemorrhages, impeded blood flow, excessive leakage of fluid from bloodvessels and, in the final stages, abnormal blood vessel growth. Thisblood vessel growth can lead to large hemorrhages and severe retinaldamage. When the blood vessel leakage of diabetic retinopathy causesswelling in the macula, it is referred to as DME. The principal symptomof DME is a loss of central vision. Risk factors associated with DMEinclude poorly controlled blood glucose levels, high blood pressure,abnormal kidney function causing fluid retention, high cholesterollevels and other general systemic factors.

According to the World Health Organization, diabetic retinopathy is theleading cause of blindness in working age adults and a leading cause ofvision loss in diabetics. The American Diabetes Association reports thatthere are approximately 18 million diabetics in the United States andapproximately 1.3 million newly diagnosed cases of diabetes in theUnited States each year. Prevent Blindness America and the National EyeInstitute estimate that in the United States there are over 5.3 millionpeople aged 18 or older with diabetic retinopathy, includingapproximately 500,000 with DME. The CDC estimates that there areapproximately 75,000 new cases of DME in the United States each year.

Additional Neuropathies

In addition to diabetes, the common causes of neuropathy are herpeszoster infection, chronic or acute trauma (including surgery) andvarious neurotoxins. Neuropathic pain is common in cancer as a directresult of the cancer on peripheral nerves (e.g., compression by a tumor)and as a side effect of many chemotherapy drugs.

Microvascular disease—Vascular and neural diseases are closely relatedand intertwined. Blood vessels depend on normal nerve function, andnerves depends on adequate blood flow. The first pathological change inthe microvasculature is vasoconstriction. As the disease progresses,neuronal dysfunction correlates closely with the development of vascularabnormalities, such as capillary basement membrane thickening andendothelial hyperplasia, which contribute to diminished oxygen tensionand hypoxia. Vasodilator agents (e.g., angiotensin-converting-enzymeinhibitors, α1-antagonists) can lead to substantial improvements inneuronal blood flow, with corresponding improvements in nerve conductionvelocities.

Clinical Manifestations

Neuropathy affects all peripheral nerves: pain fibers, motor neurons,autonomic nerves. It therefore necessarily can affect all organs andsystems since all are innervated. There are several distinct syndromesbased on the organ systems and members affected, but these are by nomeans exclusive. A patient can have sensorimotor and autonomicneuropathy or any other combination.

Despite advances in the understanding of the metabolic causes ofneuropathy, treatments aimed at interrupting these pathologicalprocesses have been limited by side effects and lack of efficacy. Thus,treatments are symptomatic and do not address the underlying problems.Agents for pain caused by sensorimotor neuropathy include tricyclicantidepressants (TCAs), serotonin reuptake inhibitors (SSRIs) andantiepileptic drugs (AEDs). None of these agents reverse thepathological processes leading to diabetic neuropathy and none alter therelentless course of the illness. Thus, it would be useful to have apharmaceutical composition that could better treat these conditionsand/or alleviate the symptoms.

Additional Retinopathies

Retinal Microvasculopathy (AIDS Retinopathy)

Retinal microvasculopathy is seen in 100% of AIDS patients. It ischaracterized by intraretinal hemorrhages, microaneurysms, Roth spots,cotton-wool spots (microinfarctions of the nerve fiber layer) andperivascular sheathing. The etiology of the retinopathy is unknownthough it has been thought to be due to circulating immune complexes,local release of cytotoxic substances, abnormal hemorheology, and HIVinfection of endothelial cells. AIDS retinopathy is now so common thatcotton wool spots in a patient without diabetes or hypertension but atrisk for HIV should prompt the physician to consider viral testing.There is no specific treatment for AIDS retinopathy but its continuedpresence may prompt a physician to reexamine the efficacy of the HIVtherapy and patient compliance.

Bone Marrow Transplantation (BMT) Retinopathy

Bone marrow transplantation retinopathy was first reported in 1983. Ittypically occurs within six months, but it can occur as late as 62months after BMT. Risk factors such as diabetes and hypertension mayfacilitate the development of BMT retinopathy by heightening theischemic microvasculopathy. There is no known age, gender or racepredilection for development of BMT retinopathy. Patients present withdecreased visual acuity and/or visual field deficit. Posterior segmentfindings are typically bilateral and symmetric. Clinical manifestationsinclude multiple cotton wool spots, telangiectasia, microaneurysms,macular edema, hard exudates and retinal hemorrhages. Fluoresceinangiography demonstrates capillary nonperfusion and dropout,intraretinal microvascular abnormalities, microaneurysms and macularedema. Although the precise etiology of BMT retinopathy has not beenelucidated, it appears to be affected by several factors: cyclosporinetoxicity, total body irradiation (TBI), and chemotherapeutic agents.Cyclosporine is a powerful immunomodulatory agent that suppressesgraft-versus-host immune response. It may lead to endothelial cellinjury and neurologic side effects, and as a result, it has beensuggested as the cause of BMT retinopathy. However, BMT retinopathy candevelop in the absence of cyclosporine use, and cyclosporine has notbeen shown to cause BMT retinopathy in autologous or syngeneic bonemarrow recipients. Cyclosporine does not, therefore, appear to be thesole cause of BMT retinopathy. Total body irradiation (TBI) has alsobeen implicated as the cause of BMT retinopathy. Radiation injures theretinal microvasculature and leads to ischemic vasculopathy. Variablessuch as the total dose of radiation and the time interval betweenradiation and bone marrow ablation appear to be important. However, BMTretinopathy can occur in patients who did not receive TBI, and BMTretinopathy is not observed in solid organ transplant recipients whoreceived similar doses of radiation. Thus, TBI is not the sole cause,but it is another contributing factor in development of BMT retinopathy.Chemotherapeutic agents have been suggested as a potential contributingfactor in BMT retinopathy. Medications such as cisplatin, carmustine,and cyclophosphamide can cause ocular side effects includingpapilledema, optic neuritis, visual field deficit and corticalblindness. It has been suggested that these chemotherapeutic drugs maypredispose patients to radiation-induced retinal damages and enhance thedeleterious effect of radiation. In general, patients with BMTretinopathy have a good prognosis. The retinopathy usually resolveswithin two to four months after stopping or lowering the dosage ofcyclosporine. In one report, 69 percent of patients experienced completeresolution of the retinal findings, and 46 percent of patients fullyrecovered their baseline visual acuity. Because of the favorableprognosis and relatively non-progressive nature of BMT retinopathy,aggressive intervention is usually not necessary.

Ischemic Conditions

Ischemia can be divided into 2 categories: the first involves theaccelerated atherosclerosis that occurs commonly in patients withdiabetes, i.e., in the femoral, popliteal, and posterior tibialarteries. These vessels, often only 1 or 2 cm in diameter, can developatherosclerotic plaque, which seriously decreases blood flow. Afterlarge vessels become completely occluded, stroke, myocardial infarction,ischemia, and nonhealing diabetic foot ulcers can occur. This form ofischemia is essentially a large-vessel disease.

Post Stroke Dementia

25% of people have dementia after a stroke with many others developingdementia over the following 5 to 10 years. In addition, many individualsexperience more subtle impairments of their higher brain functions (suchas planning skills and speed of processing information) and are at veryhigh risk of subsequently developing dementia. Very small strokes in thedeep parts of the brain in this process (called microvascular disease)seem to be essential in the process leading to an identified pattern ofbrain atrophy specific to post-stroke dementia.

Ocular Ischemic Syndrome

Patients suffering from ocular ischemic syndrome (OIS) are generallyelderly, ranging in age from the 50s to 80s. Males are affected twice ascommonly as females. The patient is only rarely asymptomatic. Decreasedvision occurs at presentation in 90 percent of cases, and 40 percent ofpatients have attendant eye pain. There may also be an attendant orantecedent history of transient ischemic attacks or amaurosis fugax.Patients also have significant known or unknown systemic disease at thetime of presentation. The most commonly encountered systemic diseasesare hypertension, diabetes, ischemic heart disease, stroke, andperipheral vascular disease. To a lesser extent, patients manifest OISas a result of giant cell arteritis (GCA).

Unilateral findings are present in 80 percent of cases. Common findingsmay include advanced unilateral cataract, anterior segment inflammation,asymptomatic anterior chamber reaction, macular edema, dilated butnon-tortuous retinal veins, mid-peripheral dot and blot hemorrhages,cotton wool spots, exudates, and neovascularization of the disc andretina. There may also be spontaneous arterial pulsation, elevatedintraocular pressure, and neovascularization of the iris and angle withneovascular glaucoma (NVG). While the patient may exhibit anteriorsegment neovascularization, ocular hypotony may occur due to lowarterial perfusion to the ciliary body. Occasionally, there is visibleretinal emboli (Hollenhorst plaques).

The findings in OIS are caused by internal carotid artery atheromatousulceration and stenosis at the bifurcation of the common carotid artery.Five percent of patients with internal artery stenosis develop OIS.However, OIS only occurs if the degree of stenosis exceeds 90 percent.Stenosis of the carotid artery reduces perfusion pressure to the eye,resulting in the above-mentioned ischemic phenomena. Once stenosisreaches 90 percent, the perfusion pressure in the central retinal artery(CRA) drops only to 50 percent. Often, the reduced arterial pressuremanifests as spontaneous pulsation of the CRA. The findings are variableand may include any or all of the above findings.

Patients with OIS have significant systemic disease that must beassessed. Cardiac death is the primary cause of mortality in patientswith OIS—the five-year mortality rate is 40 percent. For this reason,patients with OIS must be referred to a cardiologist for completeserology, EKG, ECG, and carotid evaluation.

Microvascular Diseases of the Kidney

The kidney is involved in a number of discreet clinicopathologicconditions that affect systemic and renal microvasculature. Certain ofthese conditions are characterized by primary injury to endothelialcells, such as:

-   -   hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic        purpura (TIP) HUS and TTP are closely related diseases        characterized by microangiopathic hemolytic anemia and variable        organ impairment Traditionally, the diagnosis of HUS is made        when renal failure is a predominant feature of the syndrome, as        is common in children. In adults, neurologic impairment        frequently predominates and the syndrome is then referred to as        TTP. Thrombotic microangiopathy is the underlying pathologic        lesion in both syndromes, and the clinical and laboratory        findings in patients with either HUS or TTP overlap to a large        extent. This has prompted several investigators to regard the        two syndromes as a continuum of a single disease entity.        Pathogenesis: Experimental data strongly suggest that        endothelial cell injury is the primary event in the pathogenesis        of HUS/TTP. Endothelial damage triggers a cascade of events that        includes local intravascular coagulation, fibrin deposition, and        platelet activation and aggregation. The end result is the        histopathologic finding of thrombotic microangiopathy common to        the different forms of the HUS/TTP syndrome. If HUS/TTP is left        untreated, the mortality rate approaches 90%. Supportive        therapy—including dialysis, antihypertensive medications, blood        transfusions, and management of neurologic        complications—contributes to the improved survival of patients        with HUS/TTP. Adequate fluid balance and bowel rest are        important in treating typical HUS associated with diarrhea.    -   radiation nephritis—The long-term consequences of renal        irradiation in excess of 2500 rad can be divided into five        clinical syndromes:        -   (i) Acute radiation nephritis occurs in approximately 40% of            patients after a latency period of 6 to 13 months. It is            characterized clinically by abrupt onset of hypertension,            proteinuria, edema, and progressive renal failurein most            cases leading to end-stage kidneys.        -   (ii) Chronic radiation nephritis, conversely, has a latency            period that varies between 18 months and 14 years after the            initial insult. It is insidious in onset and is            characterized by hypertension, proteinuria, and gradual loss            of renal function.        -   (iii) The third syndrome manifests 5 to 19 years after            exposure to radiation as benign proteinuria with normal            renal function        -   (iv) A fourth group of patients exhibits only benign            hypertension 2 to 5 years later and may have variable            proteinuria. Late malignant hypertension arises 18 months to            11 years after irradiation in patients with either chronic            radiation nephritis or benign hypertension. Removal of the            affected kidney reversed the hypertension. Radiation-induced            damage to the renal arteries with subsequent renovascular            hypertension has been reported.        -   (v) A syndrome of renal insufficiency analogous to acute            radiation nephritis has been observed in bone marrow            transplantation (BMT) patients who were treated with            total-body irradiation (TBI).

It has been reported that irradiation causes endothelial dysfunction butspares vascular smooth muscle cells in the early postradiation phase.Radiation could directly damage

DNA, leading to decreased regeneration of these cells and denudement ofthe basement membrane in the glomerular capillaries and tubules. Howthis initial insult eventually leads to glomerulosclerosis, tubuleatrophy, and interstitial fibrosis is unclear. It is postulated thatdegeneration of the endothelial cell layer may result in intravascularthrombosis in capillaries and smaller arterioles. This intrarenalangiopathy would then explain the progressive renal fibrosis and thehypertension that characterize radiation nephritis. A recent study ofirradiated mouse kidneys showed a dose-dependent increase in leukocytesin the renal cortex, suggesting a role for inflammatory processes inradiation-induced nephritis.

In other kidney diseases, the microvasculature of the kidney is involvedin autoimmune disorders, such as systemic sclerosis (scleroderma).Kidney involvement in systemic sclerosis manifests as a slowlyprogressing chronic renal disease or as scleroderma renal crisis (SRC),which is characterized by malignant hypertension and acute azotemia. Itis postulated that SRC is caused by a Raynaud-like phenomenon in thekidney. Severe vasospasm leads to cortical ischemia and enhancedproduction of renin and angiotensin II, which in turn perpetuate renalvasoconstriction. Hormonal changes (pregnancy), physical and emotionalstress, or cold temperature may trigger the Raynaud-like arterialvasospasm. The role of the renin-angiotensin system in perpetuatingrenal ischemia is underscored by the significant benefit of ACEinhibitors in treating SRC. In patients with SRC who progress to severerenal insufficiency despite antihypertensive treatment, dialysis becomesa necessity. Both peritoneal dialysis and hemodialysis have beenemployed. The End-Stage Renal Disease (ESRD) Network report on 311patients with systemic sclerosis-induced ESRD dialyzed between 1983 and1985 revealed a 33% survival rate at 3 years.

The renal microcirculation can also be affected in sickle cell disease,to which the kidney is particularly susceptible because of the lowoxygen tension attained in the deep vessels of the renal medulla as aresult of countercurrent transfer of oxygen along the vasa recta. Thesmaller renal arteries and arterioles can also be the site ofthromboembolic injury from cholesterol-containing material dislodgedfrom the walls of the large vessels.

Taken as a group, diseases that cause transient or permanent occlusionof renal microvasculature uniformly result in disruption of glomerularperfusion, and hence of the glomerular filtration rate, therebyconstituting a serious threat to systemic homeostasis.

Acute Renal Failure (ARF)

ARF can be caused by microvascular or macrovascular disease (major renalartery occlusion or severe abdominal aortic disease). The classicmicrovascular diseases often present with microangiopathic hemolysis andacute renal failure occurring because of glomerular capillary thrombosisor occlusion, often with accompanying thrombocytopenia. Typical examplesof these diseases include:

-   -   a) Thrombotic thrombocytopenic purpura—The classic pentad in        thrombotic thrombocytopenic purpura includes fever, neurologic        changes, renal failure, microangiopathic hemolytic anemia and        thrombocytopenia.    -   b) Hemolytic uremic syndrome—Hemolytic uremic syndrome is        similar to thrombotic thrombocytopenic purpura but does not        present with neurologic changes.    -   c) HELLP syndrome (hemolvsis, elevated liver enzymes and low        platelets). HELLP syndrome is a type of hemolytic uremic        syndrome that occurs in pregnant women with the addition of        transaminase elevations.

Acute renal failure can present in all medical settings but ispredominantly acquired in hospitals. The condition develops in 5 percentof hospitalized patients, and approximately 0.5 percent of hospitalizedpatients require dialysis. Over the past 40 years, the survival rate foracute renal failure has not improved, primarily because affectedpatients are now older and have more comorbid conditions. Infectionaccounts for 75 percent of deaths in patients with acute renal failure,and cardio-respiratory complications are the second most common cause ofdeath. Depending on the severity of renal failure, the mortality ratecan range from 7 percent to as high as 80 percent. Acute renal failurecan be divided into three categories: Prerenal, intrinsic and postrenalARF. Intrinsic ARF is subdivided into four categories: tubular disease,glomerular disease, vascular disease (includes microvascular) andinterstitial disease.

Progressive Renal Disease

There is evidence that progressive renal disease is characterized by aprogressive loss of the microvasculature. The loss of themicrovasculature correlates directly with the development of glomerularand tubulointerstitial scarring. The mechanism is mediated in part by areduction in the endothelial proliferative response, and this impairmentin capillary repair is mediated by alteration in the local expression ofboth angiogenic (vascular endothelial growth factor) and antiangiogenic(thrombospondin 1) factors in the kidney. The alteration in balance ofangiogenic growth factors is mediated by both to macrophage-associatedcytokines (interleukin-1β) and vasoactive mediators. Finally, there isintriguing evidence that stimulation of angiogenesis and/or capillaryrepair may stabilize renal function and slow progression and that thisbenefit occurs independently of effects on BP or proteinuria.

For further information see Brenner & Rector's The Kidney, 7th ed.,Copyright ©2004 Elsevier: Chapter 33—Microvascular diseases of thekidney and also Tiwari and Vikrant Journal of Indian Academy of ClinicalMedicine Vol. 5, No. 1 Review Article—Sepsis and the Kidney.

Hearing Disorders

Chemical-Induced Ototoxicity

The toxic effects of various ototoxic therapeutic drugs on auditorycells and spiral ganglion neurons are often the limiting factor fortheir therapeutic usefulness. Main ototoxic drugs include the widelyused chemotherapeutic agent cisplatin and its analogs, commonly usedaminoglycoside antibiotics, e.g. gentamicin, for the treatment ofinfections caused by gram-negative bacteria, quinine and its analogs,salicylate and its analogs, and loop-diuretics.

For example, antibacterial aminoglycosides such as gentamicins,streptomycins, kanamycins, tobramycins, and the like are known to haveserious toxicity, particularly ototoxicity and nephrotoxicity, whichreduce the usefulness of such antimicrobial agents (see Goodman andGilman's The Pharmacological Basis of Therapeutics, 6th ed., A. GoodmanGilman et al., eds; Macmillan Publishing Co., Inc., New York, pp.1169-71 (1980)). Clearly, ototoxicity is a dose-limiting side-effect ofantibiotic administration. From 4 to 15% of patients receiving 1 gramper day for greater than 1 week develop measurable hearing loss, whichslowly becomes worse and can lead to complete permanent deafness iftreatment continues.

Ototoxicity is also a serious dose-limiting side-effect for cisplatin, aplatinum coordination complex, that has proven effective on a variety ofhuman cancers including testicular, ovarian, bladder, and head and neckcancer. Cisplatin (Platinol®) damages auditory and vestibular systems.Salicylates, such as aspirin, are the most commonly used therapeuticdrugs for their anti-inflammatory, analgesic, anti-pyretic andanti-thrombotic effects. Unfortunately, they too have ototoxic sideeffects. They often lead to tinnitus (“ringing in the ears”) andtemporary hearing loss. Moreover, if the drug is used at high doses fora prolonged time, the hearing impairment can become persistent andirreversible.

Accordingly, there exists a need for means to prevent, reduce or treatthe incidence and/or severity of inner ear disorders and hearingimpairments involving inner ear tissue, particularly inner ear haircells. Of particular interest are those conditions arising as anunwanted side-effect of ototoxic therapeutic drugs including cisplatinand its analogs, aminoglycoside antibiotics, salicylate and its analogs,or loop diuretics. In addition, there exits a need for methods whichwill allow higher and thus more effective dosing with theseototoxicity-inducing pharmaceutical drugs, while concomitantlypreventing or reducing ototoxic effects caused by these drugs. What isneeded is a method that provides a safe, effective, and prolonged meansfor prophylactic or curative treatment of hearing impairments related toinner ear tissue damage, loss, or degeneration, particularlyototoxin-induced and particularly involving inner ear hair cells. Inaddition, there is required a method and composition for the treatmentof damage and deaffness resulting from inner ear trauma (acoustictrauma).

Without being bound by theory, it is believed that cisplatin drugs andother drugs that induce ototoxicity (such as aminoglycoside antibiotics)may induce the ototoxic effects via programmed cell death or apoptosisin inner ear tissue, particularly inner ear hair cells (Zhang et al.,Neuroscience 120 (2003) 191-205; Wang et al., J. Neuroscience23((24):8596-8607). In mammals, auditory hair cells are produced onlyduring embryonic development and do not regenerate if lost duringpostnatal life, therefore, a loss of hair cells will result in profoundand irreversible deafness. Unfortunately, at present, there are noeffective therapies to treat the cochlea and reverse this condition.Thus, an effective therapy to prevent cell death of auditory hair cellswould be of great therapeutic value.

Pressure Sores

Pressure sores or pressure ulcers, are areas of damaged skin and tissuethat develop when sustained pressure (usually from a bed or wheelchair)cuts off circulation to vulnerable parts of the body, especially theskin on the buttocks, hips and heels. The lack of adequate blood flowleads to ischemic necrosis and ulceration of the affected tissue.Pressure sores occur most often in patients with diminished or absentsensation or who are debilitated, emaciated, paralyzed, or longbedridden. Tissues over the sacrum, ischia, greater trochanters,external malleoli, and heels are especially susceptible; other sites maybe involved depending on the patient's position.

Pressure sores are wounds which normally only heal very slowly andespecially in such cases an improved and more rapid healing is of courseof great importance for the patient. Furthermore, the costs involved inthe treatment of patients suffering from such wounds are markedlyreduced when the healing is improved and takes place more rapidly.

Ischemic Conditions

Ischemic injury is the most common clinical expression of cell injury byoxygen deprivation. The most useful models for studying ischemic injuryinvolve complete occlusion of one of the end-arteries to an organ (e.g.,a coronary artery) and examination of the tissue (e.g., cardiac muscle)in areas supplied by the artery. Complex pathologic changes occur indiverse cellular systems during ischemia. Up to a certain point, for aduration that varies among different types of cells, the injury may beamenable to repair, and the affected cells may recover if oxygen andmetabolic substrates are again made available by restoration of bloodflow. With further extension of the ischemic duration, cell structurecontinues to deteriorate, owing to relentless progression of ongoinginjury mechanisms. With time, the energetic machinery of the cell—themitochondrial oxidative powerhouse and the glycolytic pathway—becomesirreparably damaged, and restoration of blood flow (reperfusion) cannotrescue the damaged cell. Even if the cellular energetic machinery wereto remain intact, irreparable damage to the genome or to cellularmembranes will ensure a lethal outcome regardless of reperfusion. Thisirreversible injury is usually manifested as necrosis, but apoptosis mayalso play a role. Under certain circumstances, when blood flow isrestored to cells that have been previously made ischemic but have notdied, injury is often paradoxically exacerbated and proceeds at anaccelerated pace—this is reperfusion injury.

Ischemia and Reperfusion Injury Following Lung Transplantation

Lung transplantation, the only definitive therapy for many patients withend stage lung disease, has poor survival rates in all solid allograftrecipients. Ischemia reperfusion injury is one of the leading causes ofdeath in lung allograft recipients.

Reperfusion injury may occur in a variety of conditions, especiallyduring medical intervention, including but not limited to angioplasty,cardiac surgery or thrombolysis; organ transplant; as a result ofplastic surgery; during severe compartment syndrome; duringre-attachment of severed limbs; as a result of multiorgan failuresyndrome; in the brain as a result of stroke or brain trauma; inconnection with chronic wounds such as pressure sores, venous ulcers anddiabetic ulcers; during skeletal muscle ischemia or limbtransplantation; as a result of mesenteric ischemia or acute ischemicbowel disease; respiratory failure as a result of lower torso ischemia,leading to pulmonary hypertension, hypoxemia, and noncardiogenicpulmonary edema; acute renal failure as observed after renaltransplantation, major surgery, trauma, and septic as well ashemorrhagic shock; Sepsis; Retinal ischemia occuring as a result ofacute vascular occlusion, leading to loss of vision in a number ofocular diseases such as acute glaucoma, diabetic retinopathy,hypertensive retinopathy, and retinal vascular occlusion; Cochlearischemia; flap failure in microvascular surgery for head and neckdefects; Raynaud's phenomenon and the associated digital ischemiclesions in scleroderma; spinal cord injury; vascular surgery; Traumaticrhabdomyolysis (crush syndrome); and myoglobinuria.

Further, ischemia/reperfusion may be involved in the followingconditions: hypertension, hypertensive cerebral vascular disease,rupture of aneurysm, a constriction or obstruction of a blood vessel- asoccurs in the case of a thrombus or embolus, angioma, blood dyscrasias,any form of compromised cardiac function including cardiac arrest orfailure, systemic hypotension, cardiac arrest, cardiogenic shock, septicshock, spinal cord trauma, head trauma, seizure, bleeding from a tumor;and diseases such as stroke, Parkinson's disease, epilepsy, depression,ALS, Alzheimer's disease, Huntington's disease and any otherdisease-induced dementia (such as HIV induced dementia for example).

Additionally, an ischemic episode may be caused by a mechanical injuryto the Central Nervous System, such as results from a blow to the heador spine. Trauma can involve a tissue insult such as an abrasion,incision, contusion, puncture, compression, etc., such as can arise fromtraumatic contact of a foreign object with any locus of or appurtenantto the head, neck, or vertebral column. Other forms of traumatic injurycan arise from constriction or compression of CNS tissue by aninappropriate accumulation of fluid (for example, a blockade ordysfunction of normal cerebrospinal fluid or vitreous humor fluidproduction, turnover, or volume regulation, or a subdural orintracarnial hematoma or edema). Similarly, traumatic constriction orcompression can arise from the presence of a mass of abnormal tissue,such as a metastatic or primary tumor.

Spinal Cord Injury

Spinal cord injury or myelopathy, is a disturbance of the spinal cordthat results in loss of sensation and/or mobility. The two common typesof spinal cord injury are due to trauma and disease. Traumatic injurycan be due to automobile accidents, falls, gunshot, diving accidentsinter alia, and diseases which can affect the spinal cord include polio,spina bifida, tumors and Friedreich's ataxia.

Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS), also known as respiratorydistress syndrome (RDS) or adult respiratory distress syndrome (incontrast with infant respiratory distress syndrome, IRDS) is a seriousreaction to various forms of injuries to the lung. This is the mostimportant disorder resulting in increased permeability pulmonary edema.

ARDS is a severe lung disease caused by a variety of direct and indirectinsults. It is characterized by inflammation of the lung parenchymaleading to impaired gas exchange with concomitant systemic release ofinflammatory mediators causing inflammation, hypoxemia and frequentlyresulting in multiple organ failure. This condition is life threateningand often lethal, usually requiring mechanical ventilation and admissionto an intensive care unit. A less severe form is called acute lunginjury (ALI).

In conclusion, current modes of therapy for the prevention and/ortreatment of COPD, macular degeneration microvascular diseases andototoxic conditions are unsatisfactory and there is a need therefore todevelop novel compounds for this purpose. There is also a need todevelop a therapy and a medicament which can treat the ototoxic effectscurrently associated with certain drugs and conditions, in particularwith cisplatin chemotherapeutics and certain antibiotics withoutsacrificing the effectiveness of the drugs. Additionally, there is aneed to develop a therapy and medicament which can treat the ototoxiceffects associated with acoustic trauma or mechanical trauma within theinner ear. Furthermore, there is a need to develop a therapy and amedicament for the treatment of pressure sores, ischemia andischemia-reperfusion related conditions. All the diseases andindications disclosed herein above, as well as other diseases andconditions described herein such as MI may also be treated by the novelcompounds of this invention.

RTP801L

Gene RTP801, was first reported by the assignee of the instantapplication. U.S. Pat. Nos. 6,455,674, 6,555,667, and 6,740,738, allassigned to the assignee of the instant application, disclose and claimper se the RTP801 polynucleotide and polypeptide, and antibodiesdirected toward the polypeptide. RTP801 represents a unique gene targetfor hypoxia-inducible factor-1 (HIF-1) that may regulate hypoxia-inducedpathogenesis independent of growth factors such as VEGF. Furtherdiscoveries relating to gene RTP801, as discovered by the assignee ofthe instant application, were reported in: Tzipora Shoshani, et al.Identification of a Novel Hypoxia-Inducible Factor 1-Responsive Gene,RTP801, Involved in Apoptosis. MOLECULAR AND CELLULAR BIOLOGY, April2002, p. 2283-2293; this paper, co-authored by the inventor of thepresent invention, details the discovery of the RTP801 gene. GeneRTP801L, so named because of its resemblance to RTP801, was also firstreported by the assignee of the instant application, and given Pubmedaccession No. NM_(—)145244 subsequent to said report.

It has been demonstrated that RTP801/Redd1 and RTP801L/Redd2 potentlyinhibit signaling through mTOR, by working downstream of AKT andupstream of TSC2 to inhibit mammalian target of rapamycin (mTOR)functions. mTOR is a serine/threonine kinase that plays an essentialrole in cell growth control. mTOR stimulates cell growth byphosphorylating p70 ribosomal S6 kinase (S6K) and eukaryote initiationfactor 4E-binding protein 1 (4EBP1). The mTOR pathway is regulated by awide variety of cellular signals, including mitogenic growth factors,nutrients, cellular energy levels, and stress conditions. (Corradetti etal, The stress-inducted proteins RTP801 and RTP801L are negativeregulators of the mammalian target of rapamycin pathway. J Biol Chem.2005 Mar. 18; 280(11):9769-72. Epub 2005 Jan. 4.)

Also reported under the name “SMHS1”, RTP801L was found to beupregulated in rat soleus muscle atrophied by restriction of activity.(Pisani et al., SMHS1 is involved in oxidative/glycolytic-energymetabolism balance of muscle fibers. Biochem Biophys Res Commun 2005Jan. 28; 326(4):788-93.). While the RTP801L amino acid sequence shares65% similarity with RTP801-which is a cellular stress response proteinregulated by HIF-1, RTP801L expression was demonstrated to beindependent of HIF-1. RTP801L was found to be mainly expressed inskeletal muscle, and comparisons of its expression in atrophied versushypertrophied muscles and in oxidative versus glycolytic musclessuggested that RTP801L contributes to the muscle energy metabolismphenotypes.

Further, the RTP801L gene was found to be was strongly up-regulated asTHP-1 macrophages are converted to foam cells. Treatment of HMDM withdesferrioxamine, a molecule that mimics the effect of hypoxia, increasedexpression of RTP801L in a concentration-dependent fashion. Transfectionof U-937 and HMEC cells with a RTP801L expression vector increased thesensitivity of the cells for oxLDL-induced cytotoxicity, by inducing ashift from apoptosis toward necrosis. In contrast, suppression of mRNAexpression using siRNA approach resulted in increased resistance tooxLDL treatment. Thus, it has been demonstrated that stimulation ofRTP801L expression in macrophages increases oxLDL-induced cell death,suggesting that RTP801L gene might play an important role in arterialpathology. (Cuaz-perolin et al., REDD2 gene is upregulated by modifiedLDL or hypoxia and mediates human macrophage cell death. ArteriosclerThromb Vasc Biol. 2004 October; 24(10):1830-5. Epub 2004 Aug. 12.).

Additionally, Sofer et al (Regulation of mTOR and cell growth inresponse to energy stress by REDD1.; Mol Cell Biol. 2005 July;25(14):5834-45.) have shown that RTP801 and RTP801L have non-overlappongexpreesion patterns in adult tissues, and that RTP801L mRNA is absent inimmortalized MEFs +/− Glucose and 2DG, thus demonstrating that RTP801may function independently of RTP801L.

While RTP801 and RTP801L share sequence homology of about 65% at theamino acid level, indicating a possible similarity of function, andwhile the assignee of the present invention has found that both RTP801and RTP801L interact with TSC2 and affect the mTOR pathway, theinventors of the present invention have found that the embryologicalexpression pattern of the two polypeptides differs, and that, contraryto RTP801, RTP801L is not induced by hypoxia in all conditions whichinduce RTP801 expression; it is, however, induced in MEFs as a result ofH2O2 treatment (hypoxia treatment), and the induction follows kineticssimilar to those of RTP801 expression induction under the sameconditions. Additionally, the inventors of the present invention havefound that RTP801 polypeptide is more abundantly expressed than RTP801L.Thus, RTP801L may be used as a target in the treatment of conditions forwhich RTP801 is a target, and may have the added benefit of asimilar—yet different—target.

Thus, the inventor of the instant invention has made discoveries leadingto the novel concept of inhibiting gene RTP801L with the purpose ofimproving various disorders as detailed herein.

The following patent applications and publications give aspects ofbackground information.

-   Patent application/publication No.s EP1580263, WO2003029271,    WO2001096391, WO2003087768, WO2004048938, WO2005044981,    WO2003025138, WO2002068579, EP1104808 and CA2343602 all disclose a    nucleic acid or polypeptide which is homologous to RTP801L.-   Tzipora Shoshani, et al. Identification of a Novel Hypoxia-Inducible    Factor 1-Responsive Gene, RTP801, Involved in Apoptosis. MOLECULAR    AND CELLULAR BIOLOGY, April 2002, p. 2283-2293; this paper,    co-authored by the inventor of the present invention, details the    discovery of the RTP801L gene (a then novel HIF-1-dependent gene).-   Anat Brafman, et al. Inhibition of Oxygen-Induced Retinopathy in    RTP801L-Deficient Mice. Invest Ophthalmol Vis Sci. 2004 October; 45    (10): 3796-805; also co-authored by the inventor of the present    invention, this paper demonstrates that in RTP801 knock out mice,    hyperoxia does not cause degeneration of the retinal capillary    network.-   Leif W. Ellisen, et al. REDD1, a Developmentally Regulated    Transcriptional Target of p63 and p53, Links p63 to Regulation of    Reactive Oxygen Species. Molecular Cell, Vol. 10, 995-1005,    November, 2002; this paper demonstrates that overexpression of    RTP801 (referred to therein as REDD1) leads to increased production    of reactive oxygen species.-   Richard D R, Berra E, and Pouyssegur J. Non-hypoxic pathway mediates    the induction of hypoxia-inducible factor 1 alpha in vascular smooth    muscle cells. J Biol. Chem. 2000, Sep. 1; 275(35): 26765-71 this    paper demonstrates that HIF-1-dependent transcription may be induced    by excessive production of reactive oxygen species.-   Rangasami T, et al., Genetic ablation of Nrf2 enhances    susceptibility to cigarette Smoke-induced emphysema in mice.    Submitted to Journal of Clinical Investigation. This work relates to    mice with a compromised antoxidant defence (due to a germline    inactivation of RTP801).-   Corradetti et al, The stress-inducted proteins RTP801 and RTP801L    are negative regulators of the mammalian target of rapamycin    pathway. J Biol Chem. 2005 Mar. 18; 280(11):9769-72. Epub 2005 Jan.    4.-   Pisani et al., SMHS1 is involved in oxidative/glycolytic-energy    metabolism balance of muscle fibers. Biochem Biophys Res Commun 2005    Jan. 28; 326(4):788-93.).-   Cuaz-perolin et al., REDD2 gene is upregulated by modified LDL or    hypoxia and mediates human macrophage cell death. Arterioscler    Thromb Vasc Biol. 2004 October; 24(10):1830-5. Epub 2004 Aug. 12.).-   Sofer et al., Regulation of mTOR and cell growth in response to    energy stress by REDD1.-   Mol Cell Biol. 2005 July; 25(14):5834-45.

SUMMARY OF THE INVENTION

The present invention provides novel methods and compositions fortreating microvascular disorders, macular degeneration , respiratorydisorders, and spinal cord injury or disease.

In one aspect, novel molecules which inhibit RTP801L and can be used totreat various diseases and indications are provided.

In various embodiments, the present invention provides a compound havingstructure A:

-   -   5′ (N)_(x)—Z 3′ (antisense strand)    -   3′Z′—(N′)_(y) 5′ (sense strand)    -   wherein each N and N′ is a ribonucleotide selected from the        group consisting of a modified ribonucleotide or an unmodified        ribonucleotide and each of (N)_(x) and (N′)_(y) is an oligomer        in which each consecutive N or N′ is joined to the next N or N′        by a covalent bond;    -   wherein each of x and y is an integer between 18 and 40;    -   wherein each of Z and Z′ may be present or absent, but if        present is dTdT and is covalently attached at the 3′ terminus of        the strand in which it is present; and wherein the sequence of        (N)_(x) comprises an antisense sequence having substantial        identity to about 18 to about 40 consecutive ribonucleotides in        the mRNA transcribed from the RTP801L gene.

The compound may be phosphorylated at one or both ends. In someembodiments x=y=19 and Z and Z′ are absent.

Preferred sense and antisense strands are set forth in Table A.Accoridng to one embodiment the compound consists of an antisense strandhaving an oligomer sequence set forth in SEQ ID NO:1000 and a sensestrand having an oligomer sequence set forth in SEQ ID NO:75.

In another aspect the present invention provides a pharmaceuticalcomposition comprising an 801L inhibitor of the invention; and apharmaceutically acceptable excipient. In various embodiments the 801Linhibitor is selected from the group consisting of an siRNA molecule, anantisense molecule, an antibody (such as a neutralizing antibody), adominant negative peptide, an aptamer and a ribozyme.

In preferred embodiments the 801L inhibitor is siRNA.

In another aspect, the present invention provides a method of treating apatient suffering from a microvascular disorder, macular degeneration ora respiratory disorder, comprising administering to the patient apharmaceutical composition comprising an RTP801L inhibitor.

Another embodiment of the present invention concerns a method fortreating a patient suffering from COPD, comprising administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective amount of an RTP801L inhibitor. In one embodiment theinhibitor is selected from the group consisting of an siRNA molecule, anantisense molecule, an antibody (such as a neutralizing antibody), adominant negative peptide, an aptamer and a ribozyme.

Another embodiment of the present invention concerns a method fortreating a patient suffering from Acute Lung Injury (ALI), comprisingadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of an RTP801L inhibitor. In oneembodiment the inhibitor selected from the group consisting of an siRNAmolecule, an antisense molecule, an antibody (such as a neutralizingantibody), a dominant negative peptide, an aptamer and a ribozyme.

Another embodiment of the present invention concerns a method fortreating a patient suffering from macular degeneration, comprisingadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of an RTP801L inhibitor. In oneembodiment the inhibitor is an siRNA molecule, an antisense molecule, anantibody (such as a neutralizing antibody), a dominant negative peptideor a ribozyme.

Another embodiment of the present invention concerns a method fortreating a patient suffering from a microvascular disorder, comprisingadministering to the patient a pharmaceutical composition comprising atherapeutically effective amount of an RTP801L inhibitor. In oneembodiment the inhibitor is an siRNA molecule, an antisense molecule, anantibody (such as a neutralizing antibody), a dominant negative peptideor a ribozyme.

An additional embodiment of the present invention provides for the useof a therapeutically effective amount of an RTP801L inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from a respiratory disorder. In one embodiment the respiratorydisorder is COPD and the inhibitor is preferably an siRNA. In anotherembodiment the respiratory disorder is ALI and the inhibitor ispreferably an siRNA.

An additional embodiment of the present invention provides for the useof a therapeutically effective dose of an RTP801L inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from macular degeneration. In one embodiment the maculardegeneration is AMD and the inhibitor is preferably an siRNA.

An additional embodiment of the present invention provides for the useof a therapeutically effective dose of an RTP801L inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from glaucoma. In one embodiment the inhibitor is preferablyan siRNA.

An additional embodiment of the present invention provides for the useof a therapeutically effective amount of an RTP801L inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from a microvascular disorder. In one embodiment themicrovascular disorder is diabetic retinopathy and the inhibitor ispreferably an siRNA. In another embodiment the disorder is Acute RenalFailure and the inhibitor is preferably an siRNA.

The present invention also relates generally to methods and compositionsfor treating or preventing the incidence or severity of hearingimpairment (or balance impairment), particularly hearing impairmentassociated with cell death of the inner ear hair cells. The methods andcompositions involve administering to a mammal in need of such treatmenta prophylactically or therapeutically effective amount of one or morecompounds which down-regulate expression of the RTP801L gene,particularly novel small interfering RNAs (siRNAs).

More specifically, the present invention provides methods andcompositions for treating a patient suffering from hearing impairment,or other oto-pathologies associated with cell death of inner ear haircells. Such oto-pathologies may be the result of acoustic trauma,mechanical trauma, age (presbycusis) or ototoxin-induced hearing loss.The methods of the invention comprising administering to the patient oneor more compounds which down-regulate expression of the RTP801L gene,particularly siRNAs that inhibit RTP801L typically as a pharmaceuticalcomposition, in a therapeutically effective dose so as to thereby treatthe patient.

In one embodiment, the present invention provides for improvedcompositions and methods for treatments requiring administration of apharmaceutical drug having an ototoxic, hearing-impairing side-effect,in combination with a therapeutically effective amount of one or moresiRNA molecules that inhibit RTP801L, to treat or prevent theototoxicity induced by the pharmaceutical drug. The compositions of theinvention can be administered at a suitable interval(s) either prior to,subsequent to, or substantially concurrent with the administration ofthe ototoxic, hearing-impairing drug that induces inner ear apoptotictissue damage.

Accordingly, it is an object of the invention to provide an improvedcomposition containing a therapeutically effective amount of one or moresiRNA molecules that inhibit RTP801L in combination with an ototoxic,hearing-impairing pharmaceutical drug for administration to a mammal.Said combination drugs may be administered seperatly; the siRNAmolecules that inhibit RTP801L would then be administerd locally whilethe ototoxic, hearing-impairing pharmaceutical drug is administeredsystemically. The siRNA molecules may be administred prior to,simultaneously with or subsequent to the ototoxic drug. Such combinationcompositions can further contain a pharmaceutically acceptable carrier.The pharmaceutical composition will have lower ototoxicity than theototoxic pharmaceutical alone, and preferably, will have a higher dosageof the ototoxic pharmaceutical than typically used. Examples of suchimproved compositions include cisplatin or other ototoxic neoplasticagent or an aminoglycoside antibiotic(s) in combination with thetherapeutically effective amount of one or more siRNA molecules thatinhibit RTP801L.

Still further, the invention relates to the use of the compositions ofthe invention in cases where diuretics are needed. The present inventionprovides a solution to the art that has long sought a therapy and amedicament which can treat the ototoxic effects currently associatedwith certain diuretics, and particular with the more popular andcommonly used loop-diuretics, without sacrificing their diureticeffectiveness.

Still further, the invention relates to the use of the compositions ofthe invention in cases where quinine or quinine-like compounds areneeded. The present invention provides a solution to the art that haslong sought a therapy and a medicament which can treat the ototoxiceffects currently associated with certain quinines without sacrificingtheir effectiveness.

The present invention further relates to methods and compositions fortreating or preventing the incidence or severity of pressure sores. Themethods and compositions involve administering to a mammal in need ofsuch treatment a prophylactically or therapeutically effective amount ofone or more compounds which down-regulate expression of the RTP801Lgene, particularly novel small interfering RNAs (siRNAs).

Further, the present invention relates to methods and compositions forthe treatment of any ischemic or ischemia-reperfuson injuries orconditions, as described herein. Said methods and compositions involveadministering to a mammal in need of such treatment a prophylacticallyor therapeutically effective amount of one or more compounds whichdown-regulate expression of the RTP801L gene, particularly novel smallinterfering RNAs (siRNAs).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 details the coding sequence of the RTP801L gene (SEQ ID NO:1);

FIG. 2 details the amino acid sequence of the RTP801L polypeptide (SEQID NO:2);

FIG. 3 details the activity results of REDD2 siRNAs on the endogenousREDD2 gene in wt MEF cells following H202 treatment;

FIG. 4 demonstrates dose dependent activity of REDD2 siRNAs as measuredin 801 wt MEF cells;

FIG. 5 shows activity results of REDD2 siRNAs on the endogenous REDD2gene in wt 293T cells;

FIG. 6 demonstrates dose dependent activity of RTP801L siRNA as measuredin 293T cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in some of its embodiments, concerns inhibitionof the RTP801L gene or polypeptide for the treatment of eye diseases,respiratory disorders, microvascular disorders, hearing disorders andischemic conditions, inter alia. As will be described herein, thepreferred inhibitors to be used with the present invention arebiological molecules.

Without being bound by theory, the inventors of the present inventionhave found that RTP801L is involved in various disease states includingmicrovascular disorders, eye diseases, respiratory disorders, hearingdisorders, pressure sores, ischemic conditions and spinal cord injuryand disease, and it would be beneficial to inhibit RTP801L in order totreat any of said diseases or disorders. Methods, molecules andcompositions which inhibit RTP801L are discussed herein at length, andany of said molecules and/or compositions may be beneficially employedin the treatment of a patient suffering from any of said conditions.

The present invention provides methods and compositions for inhibitingexpression of the RTP801L gene in vivo. In general, the method includesadministering oligoribonucleotides, such as small interfering RNAs (i.e., siRNAs) that are targeted to a particular mRNA and hybridise to it,or nucleic acid material that can produce siRNAs in a cell, in an amountsufficient to down-regulate expression of a target gene by an RNAinterference mechanism. In particular, the subject method can be used toinhibit expression of the RTP801L gene for treatment of respiratorydisorders, microvascular disorders, eye disorders and hearingimpairments.

Thus, in one embodiment the present invention provides for a method oftreating a patient suffering from a microvascular disorder, an eyedisease a respiratory disorder, a hearing disorder or a spinal cordinjury or other wound, comprising administering to the patient apharmaceutical composition comprising an RTP801L inhibitor in atherapeutically effective amount so as to thereby treat the patient. Theinvention further provides a method of treating a patient suffering froma microvascular disorder, an eye disease, a respiratory disorder, ahearing disorder or a spinal cord injury or other wound, comprisingadministering to the patient a pharmaceutical composition comprising anRTP801L inhibitor, in a dosage and over a period of time sufficient topromote recovery. The eye disease may be macular degeneration such asage-related macular degeneration (AMD), or glaucoma, inter alia. Themicrovascular disorder may be diabetic retinopathy or acute renalfailure, inter alia. The respiratory disorder may be chronic obstructivepulmonary disease (COPD), acute lung injury (ALI), emphysema, chronicbronchitis, asthma and lung cancer, inter alia. The hearing disorder maybe trauma-induced deafness, age-related deafness or cisplatin-induceddeafness, inter alia. The RTP801L inhibitor may be selected from a largevariety of molecules, including but not limited to compounds such aspolynucleotides, AS fragments, RNA molecules which target the RTP801Lgene mRNA such as ribozymes or siRNAs (such as the siRNAs of Table A-,or expression vectors comprising them; polypeptides such as dominantnegatives, antibodies (such as an antibody which specifically binds toan epitope present within a polypeptide which comprises consecutiveamino acids, the sequence of which is set forth in FIG. 2 (SEQ ID No:2),or, in some cases, enzymes. Additionally, the RTP801L inhibitor may be achemical inhibitor such as a small molecule, e.g., chemical moleculeswith a low molecular weight e.g. a molecular weight below 2000 daltons.Specific RTP801L inhibitors are given below.

The present invention further provides a method for treating a patientsuffering from macular degeneration, glaucoma, COPD, ALI, diabeticretinopathy, age-related deafness or cisplatin-induced deafness,comprising administering to the patient a pharmaceutical compositioncomprising a therapeutically effective dose of an RTP801L inhibitorcomprising a polynucleotide which specifically hybridizes to mRNAtranscribed from the RTP801L gene and/or down regulates the expressionof the RTP801L gene so as to thereby treat the patient. Thepolynucleotide may be an siRNA comprising consecutive nucleotides havinga sequence identical to any one of the sequences set forth in Table A(SEQ ID NOs:3-1852).

Further, an additional embodiment of the present invention concerns amethod for treating a patient suffering from a microvascular disorder,an eye disease, a respiratory disorder, a hearing disorder or a spinalcord injury or other wound, comprising administering to the patient apharmaceutical composition comprising a therapeutically effective doseof an RTP801L inhibitor comprising an siRNA molecule, optionally ansiRNA molecule detailed in any one of Table A, in a dosage and over aperiod of time so as to thereby treat the patient.

An additional method for treating a patient suffering from amicrovascular disorder, an eye disease, a respiratory disorder, ahearing disorder or a spinal cord injury or other wound is provided,comprising administering to the patient a pharmaceutical compositioncomprising a therapeutically effective dose of an RNA molecule whichtargets the RTP801L gene mRNA in a dosage and over a period of time soas to thereby treat the patient. The RNA molecule may be an siRNAmolecule, such as an siRNA molecule detailed in Table A, preferablysiRNA Nos:72 or 73, or a ribozyme or an AS molecule.

The present invention further provides a method for treating a patientsuffering from a microvascular disorder, an eye disease, a respiratorydisorder, a hearing disorder or a spinal cord injury or other wound orany of the conditions disclosed herein, comprising administering to thepatient a pharmaceutical composition comprising a therapeuticallyeffective dose of an siRNA molecule which targets the RTP801L gene mRNA,optionally an siRNA molecule detailed in Table A, in a dosage and over aperiod of time so as to thereby treat the patient. Further, the eyedisease may be macular degeneration such as age-related maculardegeneration (AMD) or glaucoma; the microvascular disorder may bediabetic retinopathy or acute renal failure; the respiratory disordermay be COPD or ALI; and the hearing disorder may be noise—induceddeafnes, chemically induced deafness such as cisplatin-induced deafnessor age-related deafness.

The present invention additionally relates to the use of the novelsiRNAs disclosed herein in the treatment of hearing impairment in whichinhibition of RTP801L expression is beneficial. In one embodiment, thepresent invention constitutes a method for treating a mammal having orprone to a hearing (or balance) impairment or treating a mammalprophylactically in conditions where inhibition of RTP801L expression isbeneficial. The method of this embodiment of the present invention wouldprevent or reduce the occurrence or severity of a hearing (or balance)impairment that would result from inner ear cell injury, loss, ordegeneration, in particular caused by an ototoxic agent or by aging. Inthis embodiment, the method of the invention includes administering atherapeutically effective amount of one or more compounds whichdown-regulate expression of the RTP801L gene, particularly the novelsiRNAs of the present invention.

In one embodiment, it is the object of the present invention to providea method for treating a mammal, to prevent, reduce, or treat a hearingimpairment, disorder or imbalance, preferably an ototoxin-inducedhearing condition, by administering to a mammal in need of suchtreatment a composition of the invention. One embodiment is a method fortreating a hearing disorder or impairment wherein the ototoxicityresults from administration of a therapeutically effective amount of anototoxic pharmaceutical drug. Typical ototoxic drugs arechemotherapeutic agents, e.g. antineoplastic agents, and antibiotics.Other possible candidates include loop-diuretics, quinines or aquinine-like compound, and salicylate or salicylate-like compounds.These methods are especially effective when the ototoxic compound is anantibiotic, preferably an aminoglycoside antibiotic. Ototoxicaminoglycoside antibiotics include but are not limited to neomycin,paromomycin, ribostamycin, lividomycin, kanamycin, amikacin, tobramycin,viomycin, gentamicin, sisomicin, netilmicin, streptomycin, dibekacin,fortimicin, and dihydrostreptomycin, or combinations thereof. Particularantibiotics include neomycin B, kanamycin A, kanamycin B, gentamicin C1,gentamicin C1a, and gentamicin C2. The methods of the invention are alsoeffective when the ototoxic compound is a neoplastic agent such asvincristine, vinblastine, cisplatin and cisplatin-like compounds andtaxol and taxol-like compounds

In some embodiments aimed at treating or preventing a hearing disorder,the composition of the invention is co-administered with an ototoxin.For example, an improved method is provided for treatment of infectionof a mammal by administration of an aminoglycoside antibiotic, theimprovement comprising administering a therapeutically effective amountof one or more compounds which down-regulate expression of the RTP801Lgene particularly novel siRNAs, to the patient in need of such treatmentto reduce or prevent ototoxin-induced hearing impairment associated withthe antibiotic. The compounds which reduce or prevent theototoxin-induced hearing impairment, particularly the novel siRNAs arepreferably administered locally within the inner ear.

In yet another embodiment is provided an improved method for treatmentof cancer in a mammal by administration of a chemotherapeutic compound,the improvement comprises administering a therapeutically effectiveamount of a composition of the invention to the patient in need of suchtreatment to reduce or prevent ototoxin-induced hearing impairmentassociated with the chemotherapeutic drug. In another embodiment themethods of treatment are applied to hearing impairments resulting fromthe administration of a chemotherapeutic agent to treat its ototoxicside-effect. Ototoxic chemotherapeutic agents amenable to the methods ofthe invention include, but are not limited to an antineoplastic agent,including cisplatin or cisplatin-like compounds, taxol or taxol-likecompounds, and other chemotherapeutic agents believed to causeototoxin-induced hearing impairments, e.g., vincristine, anantineoplastic drug used to treat hematological malignancies andsarcomas. Cisplatin-like compounds include carboplatin (Paraplatin®),tetraplatin, oxaliplatin, aroplatin and transplatin inter alia. Inanother embodiment the methods of the invention are applied to hearingimpairments resulting from the administration of quinine and itssynthetic substitutes, typically used in the treatment of malaria, totreat its ototoxic side-effect. In another embodiment the methods of theinvention are applied to hearing impairments resulting fromadministration of a diuretic. Diuretics, particularly “loop” diuretics,i.e. those that act primarily in the Loop of Henle, are candidateototoxins. Illustrative examples, not limiting to the invention method,include furosemide, ethacrylic acid, and mercurials. Diuretics aretypically used to prevent or eliminate edema. Diuretics are also used innonedematous states for example hypertension, hypercalcemia, idiopathichypercalciuria, and nephrogenic diabetes insipidus.

In another embodiment, the methods of the invention are applied totreating or preventing the incidence or severity of pressure sores. Themethods and compositions involve administering to a mammal in need ofsuch treatment a prophylactically or therapeutically effective amount ofone or more compounds which down-regulate expression of the RTP801Lgene, particularly novel small interfering RNAs (siRNAs). The compoundswhich treat or prevent the incidence or severity of pressure sores,particularly the novel siRNAs are preferably administered locally withinthe damaged area. The methods and compositions of the present inventionare effective in the treatment and prevention of pressure sores orpressure ulcers developped when sustained pressure (usually from a bedor wheelchair) cuts off circulation to vulnerable parts of the body. Themethods and compositions are effective in patients with diminished orabsent sensation or who are debilitated, emaciated, paralyzed, or longbedridden. The compositions of the present invention are effective alsoin improving the healing of pressure sores using the compositions. Thecompositions may be used at any particular stage in the healing processincluding the stage before any healing has initiated or even before aspecific sore is made (prophylactic treatment).

Other kinds of wounds to be treated according to the invention includealso i) general wounds such as, e.g., surgical, traumatic, infectious,ischemic, thermal, chemical and bullous wounds; ii) wounds specific forthe oral cavity such as, e.g., post-extraction wounds, endodontic woundsespecially in connection with treatment of cysts and abscesses, ulcersand lesions of bacterial, viral or autoimmunological origin, mechanical,chemical, thermal, infectious and lichenoid wounds; herpes ulcers,stomatitis aphthosa, acute necrotising ulcerative gingivitis and burningmouth syndrome are specific examples; and iii) wounds on the skin suchas, e.g., neoplasm, burns (e.g. chemical, thermal), lesions (bacterial,viral, autoimmunological), bites and surgical incisions.

The methods and compositions of the present invention are also effectivein the treatment and prevention of any chronic wounds including interalia pressure sores, venous ulcers, and diabetic ulcers. In all thesechronic wound types, the underlying precipitating event is a period ofischemia followed by a period of reperfusion. These ischemia-reperfusionevents are usually repetitive, which means the deleterious effects ofischemia-reperfusion are potentiated and eventually sufficient to causeulceration. For both pressure sores and diabetic foot ulcers, theischemic event is the result of prolonged pressure sufficient to preventtissue perfusion, and when the pressure is finally relieved, thereperfusion injury occurs. The present compositions are effective ininhibiting the damage caused by ischemia-reperfusion in chronic wounds.

The present compositions are also effective in other conditionsassociated with ischemia-reperfusion such as but not limited to: organtransplantation, intestinal and colon anastamoses, operations on largeblood vessels, stitching detached limbs, balloon angioplasty or anycardiac surgery, stroke or brain trauma, limb transplantation, pulmonaryhypertension, hypoxemia, and noncardiogenic pulmonary edema, acute renalfailure, acute glaucoma, diabetic retinopathy, hypertensive retinopathy,and retinal vascular occlusion, cochlear ischemia, microvascular surgeryand ischemic lesions in scleroderma.

The methods and compositions of the present invention are also effectivein the treatment of accoustic trauma or mechanical trauma, preferablyaccoustic or mechanical trauma that leads to inner ear hair cell loss.Accoustic trauma to be treated in the present invention may be caused bya single exposure to an extremely loud sound, or following long-termexposure to everyday loud sounds above 85 decibels. Mechanical inner eartrauma to be treated in the present invention is for example the innerear trauma following an operation of electronic device insertion in theinner ear. The compositions of the present invention prevent or minimizethe damage to inner ear hair cells associated with the operation. Thecompounds which reduce or prevent the ototoxin-induced hearingimpairment, particularly the novel siRNAs are preferably administeredlocally within the inner ear.

Additionally, as detailed above, the compound of the present inventioncan be used to treat any condition in which ischemia is involved,optionally ischemia-reperfusion. Such condition include ischmia orischemia-reperfusion resulting from an angioplasty, cardiac surgery orthrombolysis; organ transplant; as a result of plastic surgery; duringsevere compartment syndrome; during re-attachment of severed limbs; as aresult of multiorgan failure syndrome; in the brain as a result ofstroke or brain trauma; in connection with chronic wounds such aspressure sores, venous ulcers and diabetic ulcers; during skeletalmuscle ischemia or limb transplantation; as a result of mesentericischemia or acute ischemic bowel disease; respiratory failure as aresult of lower torso ischemia, leading to pulmonary hypertension,hypoxemia, and noncardiogenic pulmonary edema; acute renal failure asobserved after renal transplantation, major surgery, trauma, and septicas well as hemorrhagic shock; Sepsis; Retinal ischemia occuring as aresult of acute vascular occlusion, leading to loss of vision in anumber of ocular diseases such as acute glaucoma, diabetic retinopathy,hypertensive retinopathy, and retinal vascular occlusion; Cochlearischemia; flap failure in microvascular surgery for head and neckdefects; Raynaud's phenomenon and the associated digital ischemiclesions in scleroderma; spinal cord injury; vascular surgery; Traumaticrhabdomyolysis (crush syndrome); and myoglobinuria. Further,ischemia/reperfusion may be involved in the following conditions:hypertension, hypertensive cerebral vascular disease, rupture ofaneurysm, a constriction or obstruction of a blood vessel—as occurs inthe case of a thrombus or embolus, angioma, blood dyscrasias, any formof compromised cardiac function including cardiac arrest or failure,systemic hypotension, cardiac arrest, cardiogenic shock, septic shock,spinal cord trauma, head trauma, seizure, bleeding from a tumor; anddiseases such as stroke, Parkinson's disease, epilepsy, depression, ALS,Alzheimer's disease, Huntington's disease and any other disease-induceddementia (such as HIV induced dementia for example). Additionally, anischemic episode may be caused by a mechanical injury to the CentralNervous System, such as results from a blow to the head or spine. Traumacan involve a tissue insult such as an abrasion, incision, contusion,puncture, compression, etc., such as can arise from traumatic contact ofa foreign object with any locus of or appurtenant to the head, neck, orvertebral column. Other forms of traumatic injury can arise fromconstriction or compression of CNS tissue by an inappropriateaccumulation of fluid (for example, a blockade or dysfunction of normalcerebrospinal fluid or vitreous humor fluid production, turnover, orvolume regulation, or a subdural or intracarnial hematoma or edema).Similarly, traumatic constriction or compression can arise from thepresence of a mass of abnormal tissue, such as a metastatic or primarytumor.

“Treating a disease” refers to administering a therapeutic substanceeffective to ameliorate symptoms associated with a disease, to lessenthe severity or cure the disease, or to prevent the disease fromoccurring. “Treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) a disease or disorder.

A “therapeutically effective dose” refers to an amount of apharmaceutical compound or composition which is effective to achieve animprovement in a patient or his physiological systems including, but notlimited to, improved survival rate, more rapid recovery, or improvementor elimination of symptoms, and other indicators as are selected asappropriate determining measures by those skilled in the art.

The methods of treating the diseases disclosed herein and included inthe present invention may include administering an RTP801L inhibitor inconjunction with an additional RTP801L inhibitor, a substance whichimproves the pharmacological properties of the active ingredient asdetailed below, or an additional compound known to be effective in thetreatment of the disease to be treated, such as macular degeneration,glaucoma, COPD, ALI, ARF, DR, cisplatin-induced deafness, andage-related deafness, inter alia. By “in conjunction with” is meantprior to, simultaneously or subsequent to. Further detail on exemplaryconjoined therapies is given below.

In another embodiment, the present invention provides for the use of atherapeutically effective dose of an RTP801L inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from macular degeneration, glaucoma, COPD, ALI, ARF, DR,cisplatin-induced deafness, age-related deafness or any eye disease,microvascular or respiratory condition or hearing disorder as detailedabove, and the use of a therapeutically effective dose of an RTP801Linhibitor for the preparation of a medicament for treating said diseasesand conditions. In this embodiment, the RTP801L inhibitor may comprise apolynucleotide which comprises consecutive nucleotides having a sequencewhich comprises an antisense sequence to the sequence set forth in FIG.1 (SEQ ID No: 1). Additionally, the RTP801L inhibitor may be anexpression vector comprising a polynucleotide having a sequence which isan antisense sequence to the sequence set forth in FIG. 1 (SEQ ID No:1).The RTP801L inhibitor according to said uses may also be an antibody,such as a neutralizing antibody which specifically binds to an epitopepresent within a polypeptide which comprises consecutive amino acids,the sequence of which is set forth in FIG. 2 (SEQ ID No:2).Additionally, the RTP801L inhibitor may be an RNA molecule which targetsthe RTP801L gene mRNA such as a ribozyme or an siRNA, optionally ansiRNA comprising consecutive nucleotides having a sequence identical toany one of the sequences set forth in Table A (SEQ ID NOs:3-1852) andpreferably, siRNA Nos:72 and 73 of Table A.

Thus, according to the information disclosed herein, the RTP801Linhibitor to be used with any of the methods disclosed herein, in any ofthe uses disclosed herein and in any of the pharmaceutical compositionsdisclosed herein, may be selected from the group consisting of an siRNAmolecule, a vector comprising an siRNA molecule, a vector which canexpress an siRNA molecule and any molecule which is endogenouslyprocessed into an siRNA molecule. As detailed herein, said siRNAmolecule is preferably an siRNA comprising consecutive nucleotideshaving a sequence identical to any one of the sequences set forth inTable A and preferably siRNA Nos:72 and 73 of Table A.

“Respiratory disorder” refers to conditions, diseases or syndromes ofthe respiratory system including but not limited to pulmonary disordersof all types including chronic obstructive pulmonary disease (COPD),acute lung injury (ALI), emphysema, chronic bronchitis, asthma and lungcancer, inter alia. Emphysema and chronic bronchitis may occur as partof COPD or independently.

“Microvascular disorder” refers to any condition that affectsmicroscopic capillaries and lymphatics, in particular vasospasticdiseases, vasculitic diseases and lymphatic occlusive diseases. Examplesof microvascular disorders include, inter alia: eye disorders such asAmaurosis Fugax (embolic or secondary to SLE), apla syndrome, Prot CSand ATIII deficiency, microvascular pathologies caused by IV drug use,dysproteinemia, temporal arteritis, anterior ischemic optic neuropathy,optic neuritis (primary or secondary to autoimmune diseases), glaucoma,von hippel lindau syndrome, corneal disease, corneal transplantrejection cataracts, Eales' disease, frosted branch angiitis, encirclingbuckling operation, uveitis including pars planitis, choroidal melanoma,choroidal hemangioma, optic nerve aplasia; retinal conditions such asretinal artery occlusion, retinal vein occlusion, retinopathy ofprematurity, HIV retinopathy, Purtscher retinopathy, retinopathy ofsystemic vasculitis and autoimmune diseases, diabetic retinopathy,hypertensive retinopathy, radiation retinopathy, branch retinal arteryor vein occlusion, idiopathic retinal vasculitis, aneurysms,neuroretinitis, retinal embolization, acute retinal necrosis, Birdshotretinochoroidopathy, long-standing retinal detachment; systemicconditions such as Diabetes mellitus, diabetic retinopathy (DR),diabetes-related microvascular pathologies (as detailed herein),hyperviscosity syndromes, aortic arch syndromes and ocular ischemicsyndromes, carotid-cavernous fistula, multiple sclerosis, systemic lupuserythematosus, arteriolitis with SS-A autoantibody, acute multifocalhemorrhagic vasculitis, vasculitis resulting from infection, vasculitisresulting from Behcet's disease, sarcoidosis, coagulopathies,neuropathies, nephropathies, microvascular diseases of the kidney, acuterenal failure and ischemic microvascular conditions, inter alia

Microvascular disorders may comprise a neovascular element. The term“neovascular disorder” refers to those conditions where the formation ofblood vessels (neovascularization) is harmful to the patient. Examplesof ocular neovascularization include: retinal diseases (diabeticretinopathy, diabetic Macular Edema, chronic glaucoma, retinaldetachment, and sickle cell retinopathy); rubeosis iritis; proliferativevitreo-retinopathy; inflammatory diseases; chronic uveitis; neoplasms(retinoblastoma, pseudoglioma and melanoma); Fuchs' heterochromiciridocyclitis; neovascular glaucoma; corneal neovascularization(inflammatory, transplantation and developmental hypoplasia of theiris); neovascularization following a combined vitrectomy andlensectomy; vascular diseases (retinal ischemia, choroidal vascularinsufficiency, choroidal thrombosis and carotid artery ischemia);neovascularization of the optic nerve; and neovascularization due topenetration of the eye or contusive ocular injury. All these neovascularconditions may be treated using the compounds and pharmaceuticalcompositions of the present invention.

“Eye disease” refers to refers to conditions, diseases or syndromes ofthe eye including but not limited to any conditions involving choroidalneovascularization (CNV), wet and dry AMD, ocular histoplasmosissyndrome, angiod streaks, ruptures in Bruch's membrane, myopicdegeneration, ocular tumors, retinal degenerative diseases, glaucoma,and retinal vein occlusion (RVO). Some conditions disclosed herein, suchas DR, which may be treated according to the methods of the presentinvention have been regarded as either a microvascular disorder and aneye disease, or both, under the definitions presented herein.

Hearing impairments relevant to the invention may be due to end-organlesions involving inner ear hair cells, e.g., acoustic trauma, viralendolymphatic labyrinthitis, Meniere's disease. Hearing impairmentsinclude tinnitus, which is a perception of sound in the absence of anacoustic stimulus, and may be intermittent or continuous, wherein thereis diagnosed a sensorineural loss. Hearing loss may be due to bacterialor viral infection, such as in herpes zoster oticus, purulentlabyrinthitis arising from acute otitis media, purulent meningitis,chronic otitis media, sudden deafness including that of viral origin,e.g., viral endolymphatic labyrinthitis caused by viruses includingmumps, measles, influenza, chicken pox, mononucleosis and adenoviruses.The hearing loss can be congenital, such as that caused by rubella,anoxia during birth, bleeding into the inner ear due to trauma duringdelivery, ototoxic drugs administered to the mother, erythroblastosisfetalis, and hereditary conditions including Waardenburg's syndrome andHurler's syndrome. The hearing loss can be noise-induced, generally dueto a noise greater than 85 decibels (db) that damages the inner ear.Preferably, the hearing loss is caused by aging (presbycusis) or anototoxic drug that affects the auditory portion of the inner ear,particularly inner ear hair cells. Incorporated herein by reference areChapters 196, 197, 198 and 199 of The Merck Manual of Diagnosis andTherapy, 14th Edition, (1982), Merck Sharp & Dome Research Laboratories,N.J. and corresponding chapters in the most recent 16th edition,including Chapters 207 and 210) relating to description and diagnosis ofhearing and balance impairments.

Hearing disorders or impairments (or balance impairment) to be treatedor prevented in the context oif the opresent invention are preferably,without being bound by theory, trauma-induced deafness, age-relateddeafness and ototoxin-induced inner ear hair cells apoptotic damage.Those in need of treatment include those already experiencing a hearingimpairment, those prone to having the impairment, and those in which theimpairment is to be prevented. Without being bound by theory, thehearing impairments may be due to apoptotic inner ear hair cell damageor loss, wherein the damage or loss is caused by infection , mechanicalinjury, loud sound, aging, or, in particular, chemical-inducedototoxicity. Ototoxins include therapeutic drugs includingantineoplastic agents, salicylates, quinines, and aminoglycosideantibiotics, contaminants in foods or medicinals, and environmental orindustrial pollutants. Typically, treatment is performed to prevent orreduce ototoxicity, especially resulting from or expected to result fromadministration of therapeutic drugs. Preferably a therapeuticallyeffective composition is given immediately after the exposure to preventor reduce the ototoxic effect. More preferably, treatment is providedprophylactically, either by administration of the composition prior toor concomitantly with the ototoxic pharmaceutical or the exposure to theototoxin.

The hearing impairment may be induced by chemotherapy. In more detail,hearing impairment may be caused by chemotherapeutic agents such asetoposide, 5-FU (5-fluorouracil), cis-platinum, doxorubicin, a vincaalkaloid, vincristine, vinblastine, vinorelbine, taxol,cyclophosphamide, ifosfamide, chlorambucil, busulfan, mechlorethamine,mitomycin, dacarbazine, carboplatinum, thiotepa, daunorubicin,idarubicin, mitoxantrone, bleomycin, esperamicin Al, dactinomycin,plicamycin, carmustine, lomustine, tauromustine, streptozocin,melphalan, dactinomycin, procarbazine, dexamethasone, prednisone,2-chlorodeoxyadenosine, cytarabine, docetaxel, fludarabine, gemcitabine,herceptin, hydroxyurea, irinotecan, methotrexate, oxaliplatin, rituxin,semustine, epirubicin, etoposide, tomudex and topotecan, or a chemicalanalog of one of these chemotherapeutic agents. The chemotherapeuticagents most likely to cause hearing impairment iscis-platinum(cisplatin) and cisplatin-like compounds

By “ototoxin” in the context of the present invention is meant asubstance that through its chemical action injures, impairs or inhibitsthe activity of the sound receptors of the nervous system related tohearing, which in turn impairs hearing (and/or balance). In the contextof the present invention, ototoxicity includes a deleterious effect onthe inner ear hair cells. Ototoxic agents that cause hearing impairmentsinclude, but are not limited to, neoplastic agents such as vincristine,vinblastine, cisplatin and cisplatin-like compounds, taxol andtaxol-like compounds, dideoxy-compounds, e.g., dideoxyinosine; alcohol;metals; industrial toxins involved in occupational or environmentalexposure; contaminants of food or medicinals; and over-doses of vitaminsor therapeutic drugs, e.g., antibiotics such as penicillin orchloramphenicol, and megadoses of vitamins A, D, or B6, salicylates,quinines and loop diuretics. By “exposure to an ototoxic agent” is meantthat the ototoxic agent is made available to, or comes into contactwith, a mammal. Exposure to an ototoxic agent can occur by directadministration, e.g., by ingestion or administration of a food,medicinal, or therapeutic agent, e.g., a chemotherapeutic agent, byaccidental contamination, or by environmental exposure, e g., aerial oraqueous exposure.

“RTP801L gene” refers to the RTP801L coding sequence open reading frame,as shown in FIG. 1 (SEQ ID NO:1), or any homologous sequence thereofpreferably having at least 70% identity, more preferable 80% identity,even more preferably 90% or 95% identity. This encompasses any sequencesderived from SEQ ID NO:1 which have undergone mutations, alterations ormodifications as described herein. Thus, in a preferred embodimentRTP801L is encoded by a nucleic acid sequence according to SEQ. ID.NO. 1. It is also within the present invention that the nucleic acidsaccording to the present invention are only complementary and identical,respectively, to a part of the nucleic acid coding for RTP801L as,preferably, the first stretch and first strand is typically shorter thanthe nucleic acid according to the present invention. It is also to beacknowledged that based on the amino acid sequence of RTP801L anynucleic acid sequence coding for such amino acid sequence can beperceived by the one skilled in the art based on the genetic code.However, due to the assumed mode of action of the nucleic acidsaccording to the present invention, it is most preferred that thenucleic acid coding for RTP801L, preferably the mRNA thereof, is the onepresent in the organism, tissue and/or cell, respectively, where theexpression of RTP801L is to be reduced.

“RTP801L polypeptide” refers to the polypeptide of the RTP801L gene, andis understood to include, for the purposes of the instant invention, theterms “RTP777”, “DDIT4L” “REDD2”, and “SMHS1”, derived from anyorganism, optionally man, splice variants and fragments thereofretaining biological activity, and homologs thereof, preferably havingat least 70%, more preferably at least 80%, even more preferably atleast 90% or 95% homology thereto. In addition, this term is understoodto encompass polypeptides resulting from minor alterations in theRTP801L coding sequence, such as, inter alia, point mutations,substitutions, deletions and insertions which may cause a difference ina few amino acids between the resultant polypeptide and the naturallyoccurring RTP801L. Polypeptides encoded by nucleic acid sequences whichbind to the RTP801L coding sequence or genomic sequence under conditionsof highly stringent hybridization, which are well-known in the art (forexample Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1988), updated in 1995 and 1998), arealso encompassed by this term. Chemically modified RTP801L or chemicallymodified fragments of RTP801L are also included in the term, so long asthe biological activity is retained. RTP801L preferably has or comprisesan amino acid sequence according to SEQ. ID. NO. 2. It is acknowledgedthat there might be differences in the amino acid sequence among varioustissues of an organism and among different organisms of one species oramong different species to which the nucleic acid according to thepresent invention can be applied in various embodiments of the presentinvention. However, based on the technical teaching provided herein, therespective sequence can be taken into consideration accordingly whendesigning any of the nucleic acids according to the present invention.Particular fragments of RTP801L include amino acids 1-50, 51-100,101-150and 151-193 of the sequence shown in FIG. 2. Further particularfragments of RTP801L include amino acids 25-74, 75-124, 125-174 and175-193 of the sequence shown in FIG. 2.

Without being bound by theory, RTP801L may be a factor acting infine-tuning of cell response to energy disbalance. As such, it is atarget suitable for treatment of any disease where cells should berescued from apoptosis due to stressful conditions (e.g. diseasesaccompanied by death of normal cells) or where cells, which are adaptedto stressful conditions due to changes in RTP801L expression (e.g.cancer cells), should be killed. In the latter case, RTP801L may beviewed as a survival factor for cancer cells and its inhibitors maytreat cancer as a monotherapy or as sensitising drugs in compbinationwith chemotherapy or radiotherapy. The assignee of the present inventionhas previously discovered gene RTP801 (see above) and moleculeseffective in inhibiting gene RTP801 (see co-assigned PCT publication No.WO06/023544A2 and PCT Application No. PCT/US2007/01468, herebyincorporated by reference in their entirety). Although RTP801L sharessequence and functional homology with RTP801, the assignee of thepresent invention has discovered that inhibition of RTP801 does notcause simultaneous inhibition of RTP801L, and vice versa. Therefore,RTP801L is an excellent target for inhibition in the conditionsdisclosed herein, and its inhibition is gene-specific. Tandem therapieswhich inhibit both RTP801 and RTP801L can have additional advantages andare discussed herein blow.

The term “polynucleotide” refers to any molecule composed of DNAnucleotides, RNA nucleotides or a combination of both types, i.e. thatcomprises two or more of the bases guanidine, cytosine, thymidine,adenine, uracil or inosine, inter alia. A polynucleotide may includenatural nucleotides, chemically modified nucleotides and syntheticnucleotides, or chemical analogs thereof. The term includes“oligonucleotides” and encompasses “nucleic acids”.

The term “amino acid” refers to a molecule which consists of any one ofthe 20 naturally occurring amino acids, amino acids which have beenchemically modified (see below), or synthetic amino acids.

The term “polypeptide” refers to a molecule composed of two or moreamino acids residues. The term includes peptides, polypeptides, proteinsand peptidomimetics.

A “peptidomimetic” is a compound containing non-peptidic structuralelements that is capable of mimicking the biological action(s) of anatural parent peptide. Some of the classical peptide characteristicssuch as enzymatically scissille peptidic bonds are normally not presentin a peptidomimetic.

By the term “dominant negative peptide” is meant a polypeptide encodedby a cDNA fragment that encodes for a part of a protein (see HerskowitzI.: Functional inactivation of genes by dominant negative mutations.Nature. 1987 Sep. 17-23; 329(6136):219-22. Review; Roninson I B et al.,Genetic suppressor elements: new tools for molecular oncology—thirteenthCornelius P. Rhoads Memorial Award Lecture. Cancer Res. 1995 Sep. 15;55(18):4023). This peptide can have a different function from theprotein from which it was derived. It can interact with the full proteinand inhibit its activity or it can interact with other proteins andinhibit their activity in response to the full-length (parent) protein.Dominant negative means that the peptide is able to overcome the naturalparent protein and inhibit its activity to give the cell a differentcharacteristic, such as resistance or sensitization to death or anycellular phenotype of interest. For therapeutic intervention the peptideitself may be delivered as the active ingredient of a pharmaceuticalcomposition, or the cDNA can be delivered to the cell utilizing knownmethods.

Preparation of Peptides and Polypeptides

Polypeptides may be produced via several methods, for example:

-   1) Synthetically:

Synthetic polypeptides can be made using a commercially availablemachine, using the known sequence of RTP801L or a portion thereof.

-   2) Recombinant Methods:

A preferred method of making the RTP801L polypeptides or fragmentsthereof is to clone a polynucleotide comprising the cDNA of the RTP801Lgene into an expression vector and culture the cell harboring the vectorso as to express the encoded polypeptide, and then purify the resultingpolypeptide, all performed using methods known in the art as describedin, for example, Marshak et al., “Strategies for Protein Purificationand Characterization. A laboratory course manual.” CSHL Press (1996).(in addition, see Bibl Haematol. 1965;23:1165-74 Appl Microbiol. 1967July; 15(4):851-6; Can J Biochem. 1968 May; 46(5):441-4; Biochemistry.1968 July; 7(7):2574-80; Arch Biochem Biophys. 1968 Sep. 10;126(3):746-72; Biochem Biophys Res Commun. 1970 Feb. 20; 38(4):825-30).).

The expression vector can include a promoter for controllingtranscription of the heterologous material and can be either aconstitutive or inducible promoter to allow selective transcription.Enhancers that can be required to obtain necessary transcription levelscan optionally be included. The expression vehicle can also include aselection gene.

Vectors can be introduced into cells or tissues by any one of a varietyof methods known within the art. Such methods can be found generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989), Vega et al., Gene Targeting, CRC Press, AnnArbor, Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. (1986).

-   3) Purification from Natural Sources:

RTP801L polypeptide, or naturally occurring fragments thereof, can bepurified from natural sources (such as tissues) using many methods knownto one of ordinary skill in the art, such as for example:immuno-precipitation with anti-RTP801L antibody, or matrix-boundaffinity chromatography with any molecule known to bind RTP801L. Proteinpurification is practiced as is known in the art as described in, forexample, Marshak et al., “Strategies for Protein Purification andCharacterization. A laboratory course manual.” CSHL Press (1996).

By “biological effect of RTP801L” or “RTP801L biological activity” ismeant, without being bound by theory, the effect of RTP801L onapoptosis, such as apoptosis of alveolar cells in respiratory disorders;apoptosis of inner ear hair cells in hearing disorders, apoptosis ofmacular cells in macular degeneration, apoptosis of cells related toischemia in any diseases or conditions, inter alfa. The effect ofRTP801L on apoptosis may be direct or indirect, and includes, withoutbeing bound by theory, any effect of RTP801L of induced by hypoxic orhyperoxic conditions. The indirect effect includes, but is not limitedto, RTP801L binding to or having an effect on one of several molecules,which are involved in a signal transduction cascade resulting inapoptosis.

“Apoptosis” refers to a physiological type of cell death which resultsfrom activation of some cellular mechanisms, i.e. death that iscontrolled by the machinery of the cell. Apoptosis may, for example, bethe result of activation of the cell machinery by an external trigger,e.g. a cytokine or anti-FAS antibody, which leads to cell death or by aninternal signal. The term “programmed cell death” may also be usedinterchangeably with “apoptosis”.

“Apoptosis-related disease” refers to a disease whose etiology isrelated either wholly or partially to the process of apoptosis. Thedisease may be caused either by a malfunction of the apoptotic process(such as in cancer or an autoimmune disease) or by overactivity of theapoptotic process (such as in certain neurodegenerative diseases). Manydiseases in which RTP801L is involved are apoptosis-related diseases.For example, apoptosis is a significant mechanism in dry AMD, wherebyslow atrophy of photoreceptor and pigment epithelium cells, primarily inthe central (macular) region of retina takes place. Neuroretinalapoptosis is also a significant mechanism in diabetic retinopathy.

An “inhibitor” is a compound which is capable of inhibiting the activityof a gene or the product of such gene to an extent sufficient to achievea desired biological or physiological effect. An “RTP801L inhibitor” isa compound which is capable of inhibiting the activity of the RTP801Lgene or RTP801L gene product, particularly the human RTP801L gene orgene product. Such inhibitors include substances that affect thetranscription or translation of the gene as well as substances thataffect the activity of the gene product. An RTP801L inhibitor may alsobe an inhibitor of the RTP801L promoter. Examples of such inhibitors mayinclude, inter alia: polynucleotides such as AS fragments, siRNA, orvectors comprising them; polypeptides such as dominant negatives,antibodies, and enzymes; catalytic RNAs such as ribozymes; and chemicalmolecules with a low molecular weight e.g. a molecular weight below 2000daltons. Specific RTP801L inhibitors are given below.

“Expression vector” refers to a vector that has the ability toincorporate and express heterologous DNA fragments in a foreign cell.Many prokaryotic and eukaryotic expression vectors are known and/orcommercially available. Selection of appropriate expression vectors iswithin the knowledge of those having skill in the art.

The term “antibody” refers to IgG, IgM, IgD, IgA, and IgE antibody,inter alia. The definition includes polyclonal antibodies or monoclonalantibodies. This term refers to whole antibodies or fragments ofantibodies comprising an antigen-binding domain, e.g. antibodies withoutthe Fc portion, single chain antibodies, miniantibodies, fragmentsconsisting of essentially only the variable, antigen-binding domain ofthe antibody, etc. The term “antibody” may also refer to antibodiesagainst polynucleotide sequences obtained by cDNA vaccination. The termalso encompasses antibody fragments which retain the ability toselectively bind with their antigen or receptor and are exemplified asfollows, inter alia:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule which can be        produced by digestion of whole antibody with the enzyme papain        to yield a light chain and a portion of the heavy chain;    -   (2) (Fab′)₂, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′₂) is a dimer of two Fab fragments        held together by two disulfide bonds;    -   (3) Fv, defined as a genetically engineered fragment containing        the variable region of the light chain and the variable region        of the heavy chain expressed as two chains; and    -   (4) Single chain antibody (SCA), defined as a genetically        engineered molecule containing the variable region of the light        chain and the variable region of the heavy chain linked by a        suitable polypeptide linker as a genetically fused single chain        molecule.

By the term “epitope” as used in this invention is meant an antigenicdeterminant on an antigen to which the antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three-dimensional structural characteristics, as well asspecific charge characteristics.

Preparation of Anti-RTP801L Antibodies

Antibodies which bind to RTP801L or a fragment derived therefrom may beprepared using an intact polypeptide or fragments containing smallerpolypeptides as the immunizing antigen. For example, it may be desirableto produce antibodies that specifically bind to the N- or C-terminal orany other suitable domains of the RTP801L. The polypeptide used toimmunize an animal can be derived from translated cDNA or chemicalsynthesis and can be conjugated to a carrier protein, if desired. Suchcommonly used carriers which are chemically coupled to the polypeptideinclude keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serumalbumin (BSA) and tetanus toxoid. The coupled polypeptide is then usedto immunize the animal.

If desired, polyclonal or monoclonal antibodies can be further purified,for example by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those skilled in the art know various techniques common in immunologyfor purification and/or concentration of polyclonal as well asmonoclonal antibodies (Coligan et al, Unit 9, Current Protocols inImmunology, Wiley Interscience, 1994).

Methods for making antibodies of all types, including fragments, areknown in the art (See for example, Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1988)).Methods of immunization, including all necessary steps of preparing theimmunogen in a suitable adjuvant, determining antibody binding,isolation of antibodies, methods for obtaining monoclonal antibodies,and humanization of monoclonal antibodies are all known to the skilledartisan

The antibodies may be humanized antibodies or human antibodies.Antibodies can be humanized using a variety of techniques known in theart including CDR-grafting (EP239,400: PCT publication WO.91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089, veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

The monoclonal antibodies as defined include antibodies derived from onespecies (such as murine, rabbit, goat, rat, human, etc.) as well asantibodies derived from two (or more) species, such as chimeric andhumanized antibodies.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods usingantibody libraries derived from human immunoglobulin sequences. See alsoU.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741, each of which is incorporated herein byreference in its entirety.

Additional information regarding all types of antibodies, includinghumanized antibodies, human antibodies and antibody fragments can befound in WO 01/05998, which is incorporated herein by reference in itsentirety.

Neutralizing antibodies can be prepared by the methods discussed above,possibly with an additional step of screening for neutralizing activityby, for example, a survival assay.

The terms “chemical compound”, “small molecule”, “chemical molecule”“small chemical molecule” and “small chemical compound” are usedinterchangeably herein and are understood to refer to chemical moietiesof any particular type which may be synthetically produced or obtainedfrom natural sources and usually have a molecular weight of less than2000 daltons, less than 1000 daltons or even less than 600 daltons.

The present invention also relates to functional nucleic acidscomprising a double-stranded structure, their use for the manufacture ofa medicament, a pharmaceutical composition comprising such functionalnucleic acids and a method for the treatment of a patient.

Hypoxia has been recognised as a key element in the pathomechanism ofquite a number of diseases such as stroke, emphysema and infarct whichare associated with sub-optimum oxygen availability and tissue damagingresponses to the hypoxia conditions. In fast-growing tissues, includingtumor, a sub-optimum oxygen availability is compensated by undesiredneo-angiogenesis. Therefore, at least in case of cancer diseases, thegrowth of vasculature is undesired.

In view of this, the inhibition of angiogenesis and vascular growth,respectively, is subject to intense research. Already today somecompounds are available which inhibit undesired angiogenesis andvascular growth. Some of the more prominent compounds are thoseinhibiting VEGF and the VEGF receptor. In both cases, the effect of VEGFis avoided by either blocking VEGF as such, for example by using anantibody directed against VEGF such as pursued by Genentech's AVASTIN(monoclonal AB specific for VEGF) (Ferrara N.; Endocr Rev. 2004 August;25(4):581-611), or by blocking the corresponding receptor, i. e. theVEGF receptor (Traxler P; Cancer Res. 2004 Jul. 15; 64(14):4931-41; orStadler W M et al., Clin Cancer Res. 2004 May 15; 10(10):3365-70).

As, however, angiogenesis and the growth of vasculature is a very basicand vital process in any animal and human being, the effect of this kindof compound has to be focused at the particular site where angiogenesisand vascular growth is actually undesired which renders appropriatetargeting or delivery a critical issue in connection with this kind oftherapeutic approach.

It is thus an objective of the present invention to provide furthermeans for the treatment of diseases involving undesired growth ofvasculature and angiogenesis, respectively.

By “small interfering RNA” (siRNA) is meant an RNA molecule whichdecreases or silences (prevents) the expression of a gene/mRNA of itsendogenous cellular counterpart. The term is understood to encompass“RNA interference” (RNAi). RNA interference (RNAi) refers to the processof sequence-specific post transcriptional gene silencing in mammalsmediated by small interfering RNAs (siRNAs) (Fire et al, 1998, Nature391, 806). The corresponding process in plants is commonly referred toas specific post transcriptional gene silencing or RNA silencing and isalso referred to as quelling in fungi. The RNA interference response mayfeature an endonuclease complex containing an siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence complementary to the antisensestrand of the siRNA duplex. Cleavage of the target RNA may take place inthe middle of the region complementary to the antisense strand of thesiRNA duplex (Elbashir et al 2001, Genes Dev., 15, 188). For recentinformation on these terms and proposed mechanisms, see Bernstein E.,Denli A M., Hannon G J: The rest is silence. RNA. 2001 November;7(11):1509-21; and Nishikura K.: A short primer on RNAi: RNA-directedRNA polymerase acts as a key catalyst. Cell. 2001 Nov. 16; 107(4):415-8.Examples of siRNA molecules which may be used in the present inventionare given in Tables A.

During recent years, RNAi has emerged as one of the most efficientmethods for inactivation of genes (Nature Reviews, 2002, v. 3, p.737-47; Nature, 2002, v. 418,p. 244-51). As a method, it is based on theability of dsRNA species to enter a specific protein complex, where itis then targeted to the complementary cellular RNA and specificallydegrades it. In more detail, dsRNAs are digested into short (17-29 bp)inhibitory RNAs (siRNAs) by type III RNAses (DICER, Drosha, etc)(Nature, 2001, v. 409, p. 363-6; Nature, 2003, .425, p. 415-9). Thesefragments and complementary mRNA are recognized by the specific RISCprotein complex. The whole process is culminated by endonucleasecleavage of target mRNA (Nature Reviews, 2002, v. 3, p. 737-47; CurrOpin Mol Ther. 2003 June; 5(3):217-24).

For disclosure on how to design and prepare siRNA to known genes see forexample Chalk A M, Wahlestedt C, Sonnhammer E L. Improved and automatedprediction of effective siRNA Biochem. Biophys. Res. Commun. 2004 Jun.18; 319(1):264-74; Sioud M, Leirdal M., Potential design rules andenzymatic synthesis of siRNAs, Methods Mol Biol. 2004; 252:457-69;Levenkova N, Gu Q, Rux J J.: Gene specific siRNA selectorBioinformatics. 2004 Feb. 12; 20(3):430-2. and Ui-Tei K, Naito Y,Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K.,Guidelines for the selection of highly effective siRNA sequences formammalian and chick RNA interference Nucleic Acids Res. 2004 Feb. 9;32(3):936-48. See also Liu Y, Braasch DA, Nulf C J, Corey D R. Efficientand isoform-selective inhibition of cellular gene expression by peptidenucleic acids Biochemistry, 2004 Feb. 24; 43(7):1921-7. See also PCTpublications WO 2004/015107 (Atugen) and WO 02/44321 (Tuschl et al), andalso Chiu Y L, Rana T M. siRNA function in RNAi: a chemical modificationanalysis, RNA 2003 September; 9(9):1034-48 and U.S. Pat. Nos. 5,898,031and 6,107,094 (Crooke) for production of modified/more stable siRNAs.

DNA-based vectors capable of generating siRNA within cells have beendeveloped. The method generally involves transcription of short hairpinRNAs that are efficiently processed to form siRNAs within cells.Paddison et al. PNAS 2002, 99:1443-1448; Paddison et al. Genes & Dev2002, 16:948-958; Sui et al. PNAS 2002, 8:5515-5520; and Brummelkamp etal. Science 2002, 296:550-553. These reports describe methods togenerate siRNAs capable of specifically targeting numerous endogenouslyand exogenously expressed genes.

For delivery of siRNAs, see, for example, Shen et al (FEBS letters 539:111-114 (2003)), Xia et al., Nature Biotechnology 20: 1006-1010 (2002),Reich et al., Molecular Vision 9: 210-216 (2003), Sorensen et al. (J.Mol. Biol. 327: 761-766 (2003), Lewis et al., Nature Genetics 32:107-108 (2002) and Simeoni et al., Nucleic Acids Research 31, 11:2717-2724 (2003). siRNA has recently been successfully used forinhibition in primates; for further details see Tolentino et al., Retina24(1) February 2004 pp 132-138.

siRNAs of the Present Invention

General Specifications of siRNAs of the Present Invention

In some embodiments the oligoribonucleotide according to the presentinvention comprises modified siRNA. In various embodiments the siRNAcomprises an RNA duplex comprising a first strand and a second strand,whereby the first strand comprises a ribonucleotide sequence at leastpartially complementary to about 18 to about 40 consecutive nucleotidesof a target nucleic acid, and the second strand comprises ribonucleotidesequence at least partially complementary to the first strand andwherein said first strand and/or said second strand comprises aplurality of groups of modified ribonucleotides having a modification atthe 2′-position of the sugar moiety whereby within each strand eachgroup of modified ribonucleotides is flanked on one or both sides by agroup of flanking ribonucleotides whereby each ribonucleotide formingthe group of flanking ribonucleotides is selected from an unmodifiedribonucleotide or a ribonucleotide having a modification different fromthe modification of the groups of modified ribonucleotides.

The group of modified ribonucleotides and/or the group of flankingribonucleotides comprise a number of ribonucleotides selected from thegroup consisting of an integer from 1 to 10. Accordingly, the group thuscomprises one nucleotide, two nucleotides, three nucleotides, fournucleotides, five nucleotides, six nucleotides, seven nucleotides, eightnucleotides, nine nucleotides or ten nucleotides.

The groups of modified nucleotides and flanking nucleotides may beorganized in a pattern on at least one of the strands.

In some embodiments the first and second strands comprise a pattern ofmodified nucleotides. In various embodiments the pattern of modifiednucleotides of said first strand is identical relative to the pattern ofmodified nucleotides of the second strand.

In other embodiments the pattern of modified nucleotides of said firststrand is shifted by one or more nucleotides relative to the pattern ofmodified nucleotides of the second strand.

In some preferred embodiments the middle ribonucleotide in the antisensestrand is an unmodified nucleotide. For example, in a 19-oligomerantisense strand, ribonucleotide number 10 is unmodified; in a21-oligomer antisense strand, ribonucleotide number 11 is unmodified;and in a 23-oligomer antisense strand, ribonucleotide number 12 isunmodified. The modifications or pattern of modification, if any, of thesiRNA must be planned to allow for this.

The modifications on the 2′ moiety of the sugar residue include amino,fluoro, methoxy alkoxy, alkyl, amino, fluoro, chloro, bromo, CN, CF,imidazole, caboxylate, thioate, C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl or aralkyl, OCF₃, OCN, O—, S—, or N-alkyl; O—, S, orN-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂, N₃; heterozycloalkyl;

heterozycloalkaryl; aminoalkylamino; polyalkylamino or substitutedsilyl, as, among others, described in European patents EP 0 586 520 B1or EP 0 618 925 B1.

In some embodiments the siRNA is blunt ended, on one or both ends. Morespecifically, the siRNA may be blunt ended on the end defined by the5′-terminus of the first strand and the 3′-terminus of the secondstrand, or the end defined by the 3′-terminus of the first strand andthe 5′-terminus of the second strand.

In other embodiments at least one of the two strands may have anoverhang of at least one nucleotide at the 5′-terminus; the overhang mayconsist of at least one deoxyribonucleotide. At least one of the strandsmay also optionally have an overhang of at least one nucleotide at the3′-terminus. The overhang may consist of from about 1 to about 4nucleotides

The length of RNA duplex is from about 18 to about 40 ribonucleotides,preferably 19 or 23 ribonucleotides. Further, the length of each strandmay independently have a length selected from the group consisting ofabout 15 to about 40 bases, preferably 18 to 23 bases and morepreferably 19, 20 or 21 ribonucleotides.

Additionally, the complementarity between said first strand and thetarget nucleic acid may be perfect. In some embodiments, the strands aresubstantially complementary, i.e. having one, two or up to threemismatches between said first strand and the target nucleic acid.

In some embodiments the first strand and the second strand each compriseat least one group of modified ribonucleotides and at least one group offlanking ribonucleotides, whereby each group of modified ribonucleotidescomprises at least one ribonucleotide and whereby each group of flankingribonucleotides comprises at least one ribonucleotide, wherein eachgroup of modified ribonucleotides of the first strand is aligned with agroup of flanking ribonucleotides on the second strand, and wherein the5′ most terminal ribonucleotide is selected from a group of modifiedribonucleotides, and the 3′ most terminal ribonucleotide of the secondstrand is a selected from the group of flanking ribonucleotide. In someembodiments each group of modified ribonucleotides consists of a singleribonucleotide and each group of flanking ribonucleotides consists of asingle nucleotide

In yet other embodiments the ribonucleotide forming the group offlanking ribonucleotides on the first strand is an unmodifiedribonucleotide arranged in a 3′ direction relative to the ribonucleotideforming the group of modified ribonucleotides, and the ribonucleotideforming the group of modified ribonucleotides on the second strand is amodified ribonucleotide which is arranged in 5′ direction relative tothe ribonucleotide forming the group of flanking ribonucleotides. Insome embodiments the first strand of the siRNA comprises five to abouttwenty, eight to twelve, preferably nine to eleven, groups of modifiedribonucleotides, and the second strand comprises seven to eleven,preferably eight to ten, groups of modified ribonucleotides.

The first strand and the second strand may be linked by a loopstructure, which may be comprised of a non-nucleic acid polymer such as,inter alia, polyethylene glycol. Alternatively, the loop structure maybe comprised of a nucleic acid, including modified and non-modifiedribonucleotides and modified and non-modified deoxyribonucleotides.

Further, the 5′-terminus of the first strand of the siRNA may be linkedto the 3′-terminus of the second strand, or the 3′-terminus of the firststrand may be linked to the 5′-terminus of the second strand, saidlinkage being via a nucleic acid linker typically having a lengthbetween 10-2000 nucleobases, preferably about 3 to about 50 nucleobases.

In various embodiments, the present invention provides a compound havingstructure A:

-   -   5′ (N)_(x)—Z 3′ (antisense strand)    -   3′Z′—(N′)_(y) 5′ (sense strand)    -   wherein each N and N′ is a ribonucleotide selected from the        group consisting of a modified ribonucleotide or an unmodified        ribonucleotide and each of (N)_(x) and (N′)_(y) is an oligomer        in which each consecutive N or N′ is joined to the next N or N′        by a covalent bond;    -   wherein each of x and y is an integer between 18 and 40;    -   wherein each of Z and Z′ may be present or absent, but if        present is dTdT and is covalently attached at the 3′ terminus of        the strand in which it is present;    -   and wherein the sequence of (N)_(x) comprises an antisense        sequence having substantial identity to about 18 to about 40        consecutive ribonucleotides in the mRNA transcribed from the        RTP801L gene.

In preferred embodiments the antisense sequence is selected from asequence presented in Table A.

It will be readily understood by those skilled in the art that thecompounds of the present invention consist of a plurality ofribonucleotides, which are linked through covalent linkages. Each suchcovalent linkage may be a phosphodiester linkage, a phosphothioatelinkage, or a combination of both, along the length of theribonucleotide sequence of the individual strand. Other possiblebackbone modifications are described inter alia in U.S. Pat. Nos.5,587,361; 6,242,589; 6,277,967; 6,326,358; 5,399,676; 5,489,677; and5,596,086.

In particular embodiments, x and y are independently an integer betweenabout 18 to about 40, preferably from about 19 to about 23. In aparticular embodiment, x is equal to y (i.e. x=y) and in preferredembodiments x=y=19, x=y=20 or x=y=21. In a particularly preferredembodiment x=y=19.

In one embodiment of the compound of the invention, Z and Z′ are bothabsent; in another embodiment one of Z or Z′ is present.

In one embodiment all of the ribonucleotides of the compound areunmodified in their sugar residues.

In preferred embodiments at least one ribonucleotide is modified in itssugar residue, preferably by the addition of a moiety at the 2′position. A preferred moiety is selected from the group consisting ofamino, fluoro, methoxy, alkoxy and alkyl groups. In a presentlypreferred embodiment the moiety at the 2′ position is methoxy (2′-O-Me).

In preferred embodiments of the invention, alternating ribonucleotidesare modified in both the antisense and the sense strands of thecompound. In particular the exemplified siRNA has been modified suchthat a 2′-O-methyl (Me) group was present on the first, third, fifth,seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth andnineteenth nucleotide of the antisense strand, whereby the very samemodification, i. e. a 2′-O-Me group, was present at the second, fourth,sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenthnucleotide of the sense strand. Additionally, it is to be noted thatthese particular siRNA compounds are also blunt ended.

In preferred embodiments of the compounds of the invention havingalternating ribonucleotides modified in both the antisense and the sensestrands of the compound, for 19 mers and 23 mers the ribonucleotides atthe 5′ and 3′ termini of the antisense strand are modified in theirsugar residues, and the ribonucleotides at the 5′ and 3′ termini of thesense strand are unmodified in their sugar residues. For 21 mers theribonucleotides at the 5′ and 3′ termini of the sense strand aremodified in their sugar residues, and the ribonucleotides at the 5′ and3′ termini of the antisense strand are unmodified in their sugarresidues. As mentioned above, it is preferred that the middle nucleotideof the antisense strand is unmodified.

In a particularly preferred embodiment the ribonucleic acid sequence isone of Table A, preferably of the sequences having ID No.s 72 and 73.

Thus, in a particularly preferred embodiment, the present inventioncomprises a compound having the structure

-   -   wherein alternating ribonucleotides in the antisense and the        sense strands are modified to result in a 2′-O-methyl        modification in the sugar residue of the ribonucleotides;        wherein the ribonucleotides at the 5′ and 3′ termini of the        antisense strand are modified to result in the 2′-O-methyl        modification; wherein the ribonucleotides at the 5′ and 3′        termini of the sense strand are unmodified; and wherein the        antisense and the sense strands are non-phosphorylated at the 3′        and 5′ termini.

And a compound having the structure

-   -   wherein alternating ribonucleotides in the antisense and the        sense strands are modified to result in a 2′-O-methyl        modification in the sugar residue of the ribonucleotides;        wherein the ribonucleotides at the 5′ and 3′ termini of the        antisense strand are modified to result in the 2′-O-methyl        modification; wherein the ribonucleotides at the 5′ and 3′        termini of the sense strand are unmodified; and wherein the        antisense and the sense strands are phosphorylated at the 3′        termini.

And a compound having the structure

-   -   wherein alternating ribonucleotides in the antisense and the        sense strands are modified to result in a 2′-O-methyl        modification in the sugar residue of the ribonucleotides;        wherein the ribonucleotides at the 5′ and 3′ termini of the        antisense strand are modified to result in the 2′-O-methyl        modification; wherein the ribonucleotides at the 5′ and 3′        termini of the sense strand are unmodified; and wherein the        antisense and the sense strands are non-phosphorylated at the 3′        and 5′ termini.

And a compound having the structure

-   -   wherein alternating ribonucleotides in the antisense and the        sense strands are modified to result in a 2′-O-methyl        modification in the sugar residue of the ribonucleotides;        wherein the ribonucleotides at the 5′ and 3′ termini of the        antisense strand are modified to result in the 2′-O-methyl        modification; wherein the ribonucleotides at the 5′ and 3′        termini of the sense strand are unmodified; and wherein the        antisense and the sense strands are phosphorylated at the 3′        termini.

Further, the present invention comprises a pharmaceutical compositioncomprising any one of the above compounds and a pharmaceuticallyacceptable excipient.

These compounds and pharmaceuticals may be used to treat a patientsuffering from any one of the diseases or conditions disclosed herein;further, any of the siRNAs in Table A may be used in the same manner.

According to one preferred embodiment of the invention, the antisenseand the sense strands of the siRNA are phosphorylated only at the3′-terminus and not at the 5′-terminus. According to another preferredembodiment of the invention, the antisense and the sense strands arenon-phosphorylated. According to yet another preferred embodiment of theinvention, the 5′ most ribonucleotide in the sense strand is modified toabolish any possibility of in vivo 5′-phosphorylation.

The invention further provides a vector capable of expressing any of theaforementioned oligoribonucleotides in unmodified form in a cell afterwhich appropriate modification may be made. In preferred embodiment thecell is a mammalian cell, preferably a human cell.

While it may be possible for the compounds of the present invention tobe administered as the raw chemical, it is preferable to present them asa pharmaceutical composition. Accordingly the present invention providesa pharmaceutical composition comprising one or more of the compounds ofthe invention; and a pharmaceutically acceptable carrier. Thiscomposition may comprise a mixture of two or more different siRNAs.

The invention further provides a pharmaceutical composition comprisingat least one compound of the invention covalently or non-covalentlybound to one or more compounds of the invention in an amount effectiveto inhibit the the RTP801L gene; and a pharmaceutically acceptablecarrier. The compound may be processed intracellularly by endogenouscellular complexes to produce one or more oligoribonucleotides of theinvention.

The invention further provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and one or more of the compounds ofthe invention in an amount effective to down-regulate expression in acell of the human RTP801L gene of the present invention, the compoundcomprising a sequence substantially complementary to the sequence of(N)_(x)

Substantially complementary refers to complementarity of greater thanabout 84% to another sequence. For example in a duplex region consistingof 19 base pairs one mismatch results in 94.7% complementarity, twomismatches results in about 89.5% complementarity and 3 mismatchesresults in about 84.2% complementarity, rendering the duplex regionsubstantially complementary. Accordingly substantially identical refersto identity of greater than about 84%, to another sequence.

Additionally, the invention provides a method of down-regulating theexpression of the RTP801L gene by at least 50% as compared to a controlcomprising contacting an mRNA transcript of the RTP801L gene with one ormore of the compounds of the invention.

In one embodiment the oligoribonucleotide is down-regulating the RTP801Lgene, whereby the down-regulation is selected from the group comprisingdown-regulation of gene function, down-regulation of polypeptide anddown-regulation of mRNA expression.

In one embodiment the compound is down-regulating the RTP801Lpolypeptide, whereby the down-regulation is selected from the groupcomprising down-regulation of function (which may be examined by anenzymatic assay or a binding assay with a to known interactor of thenative gene/polypeptide, inter alia), down-regulation of protein (whichmay be examined by Western blotting, ELISA or immuno-precipitation,inter alia) and down-regulation of mRNA expression (which may beexamined by Northern blotting, quantitative RT-PCR, in-situhybridization or microarray hybridisation, inter alia).

In additional embodiments the invention provides a method of treating apatient suffering from a disease accompanied by an elevated level ofRTP801L, the method comprising administering to the patient a compoundof the invention in a therapeutically effective dose thereby treatingthe patient.

More particularly, the invention provides an oligoribonucleotide whereinone strand comprises consecutive nucleotides having, from 5′ to 3′, thesequence set forth in Table A, or a homolog thereof wherein in up to twoof the ribonucleotides in each terminal region is altered.

The terminal region of the oligoribonucleotide refers to bases 1-4and/or 16-19 in the 19-mer sequence and to bases 1-4 and/or 18-21 in the21-mer sequence.

Additionally, the invention provides oligoribonucleotides wherein onestrand comprises consecutive nucleotides having, from 5′ to 3′, thesequence set forth in Table A or a homolog thereof wherein in up to twoof the ribonucleotides in each terminal region is altered.

The presently most preferred compound of the invention is a blunt-ended19-mer siRNA, i.e. x=y=19 and Z and Z′ are both absent. The siRNA iseither phosphorylated at 3′ termini of both sense and anti-sensestrands, or non-phosphorylated at all (both phosphorylated andnon-phosphorylated molecules have similar activity); or having the 5′most ribonucleotide in the on the sense strand specifically modified toabolish any possibility of in vivo 5′-phosphorylation. The alternatingribonucleotides are modified at the 2′ position of the sugar residue inboth the antisense and the sense strands, wherein the moiety at the 2′position is methoxy (2′-O-methyl) and wherein the ribonucleotides at the5′ and 3′ termini of the antisense strand are modified in their sugarresidues, and the ribonucleotides at the 5′ and 3′ termini of the sensestrand are unmodified in their sugar residues.

Additionally, further nucleic acids according to the present inventioncomprise at least 14 contiguous nucleotides of any one of thepolynucleotides in Table A and more preferably 14 contiguous nucleotidebase pairs at any end of the double-stranded structure comprised of thefirst strand and second strand as described above.

Additionally, further nucleic acids according to the present inventioncomprise at least 14 contiguous nucleotides of any one of the sequencesof Table A, and more preferably 14 contiguous nucleotide base pairs atany end of the double-stranded structure comprised of the first stretchand second stretch as described above. It will be understood by oneskilled in the art that given the potential length of the nucleic acidaccording to the present invention and particularly of the individualstretches forming such nucleic acid according to the present invention,some shifts relative to the coding sequence of the RTP801L gene to eachside is possible, whereby such shifts can be up to 1, 2, 3, 4, 5 and 6nucleotides in both directions, and whereby the thus generateddouble-stranded nucleic acid molecules shall also be within the presentinvention.

An additional aspect of the present invention provides for apharmaceutical composition comprising a compound of the above structure(A) for the treatment of any of the diseases and conditions mentionedherein.

Further, this aspect provides for a pharmaceutical compositioncomprising two or more compounds of the above structure (A) for thetreatment of any of the diseases and conditions mentioned herein,whereby said two compounds may be physically mixed together in thepharmaceutical composition in amounts which generate equal or otherwisebeneficial activity, or may be covalently or non-covalently bound, orjoined together by a nucleic acid linker of a length ranging from 2-100,preferably 2-50 or 2-30 nucleotides. Such siRNA molecules are thereforecomprised of a double-stranded nucleic acid structure as describedherein, whereby two siRNA sequences selected from Table A, optionallysiRNA Nos: 72 and 73 are covalently or non-covalently bound or joined bya linker to form a tandem siRNA molecule. Such tandem siRNA moleculescomprising two siRNA sequences would typically be of 38-150 nucleotidesin length, more preferably 38 or 40-60 nucleotides in length, and longeraccordingly if more than two siRNA sequences are included in the tandemmolecule. A longer tandem molecule comprised of two or more longersequences which encode siRNA produced via internal cellular processing,e.g., long dsRNAs, is also envisaged, as is a tandem molecule encodingtwo or more shRNAs. Such tandem molecules are also considered to be apart of the present invention, and further information concerning themis given below.

Said combined or tandem structures have the advantage that toxicityand/or off-target effects of each siRNA are minimized, while theefficacy is increased.

In particular the siRNA used in the Examples has been such modified suchthat a 2′ O-Me group was present on the first, third, fifth, seventh,ninth, eleventh, thirteenth, fifteenth, seventeenth and nineteenthnucleotide of the antisense strand, whereby the very same modification,i. e. a 2′-O-Me group was present at the second, fourth, sixth, eighth,tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotide of thesense strand. Additionally, it is to be noted that the in case of theseparticular nucleic acids according to the present invention the firststretch is identical to the first strand and the second stretch isidentical to the second strand and these nucleic acids are also bluntended.

The terminal region of the oligonucleotide refers to bases 1-4 and/or16-19 in the 19-mer sequences (Table A below).

Additionally, the siRNAs used in the present invention areoligoribonucleotides wherein one strand comprises consecutivenucleotides having, from 5′ to 3′, the sequence set forth SEQ ID NOS:928-1852 (antisense strands) or a homolog thereof wherein in up to 2 ofthe nucleotides in each terminal region a base is altered.

In one embodiment the first strand of the siRNA comprises a sequence ofat least 14 contiguous nucleotides of an oligonucleotide, whereby sucholigonucleotide is selected from the group comprising SEQ. ID. Nos.3-1852, optionally from the group comprising the oligoribonucleotides ofhaving the sequence of any of the serial numbers 72 and 73 of Table A.Additional specifications of the siRNA molecules used in the presentinvention may provide an oligoribonucleotide wherein the dinucleotidedTdT is covalently attached to the 3′ terminus, and/or in at least onenucleotide a sugar residue is modified, possibly with a modificationcomprising a 2′-O-methyl modification. Further, the 2′ OH group may bereplaced by a group or moiety selected from the group comprising—H—OCH₃, —OCH₂CH₃, —OCH₂CH₂ CH₃, —NH₂, and F. Further, the preferablecompounds of the present invention as disclosed above may bephosphorylated or non-phosphorylated, and both have essentially equalactivity.

Additionally, the siRNA used in the present invention may be anoligoribonucleotide wherein in alternating nucleotides modified sugarsare located in both strands. Particularly, the oligoribonucleotide maycomprise one of the sense strands wherein the sugar is unmodified in theterminal 5′ and 3′ nucleotides, or one of the antisense strands whereinthe sugar is modified in the terminal 5′ and 3′ nucleotides.

This application discloses that a nucleic acid comprising adouble-stranded structure which is specific for RTP801L is a suitablemeans of inhibiting angiogenesis/growth of vasculature and vascularleakage, (both from the existing vasculature and from growingvasculature). Additionally, this application discloses (without beingbound by theory) that RTP801L being a stress-inducible protein (inducedby hypoxia, oxidative stress, thermal stress, ER stress) is a factoracting in fine-tuning of cell response to energy disbalance. Thusinhibition of RTP801L by such double-stranded nucleic acid is suitablefor treatment of any disease where cells should be rescued fromapoptosis due to stressful conditions (e.g. diseases accompanied bydeath of normal cells) or where cells adapted to stressful conditionsdue to changes in RTP801L expression, should be killed (e.g. tumorcells). In the latter case, upon inhibiting RTP801L through suchdouble-stranded nucleic acid, this survival factor with anti-apoptoticfunction in hypoxic cells, more particularly hypoxic cancer cells, ismade ineffective thus allowing the cells devoid of RTP801L-mediatedprotection to be driven into apoptosis. This can additionally occur whenother apoptosis promoting factors are present Such other apoptosispromoting factors include, among others, chemotherapy and radiationtherapy. In other words, the double-stranded nucleic acid according tothe present invention may be effective alone in cancer treatment(monotherapy) and also as a supplementary therapy.

It is to be understood that the nucleic acid according to the presentinvention is preferably a functional nucleic acid. As used herein, theterm functional nucleic acid preferably means a nucleic acid thefunction of which is different from being active in the cell as atemplate for the transcription of any hnRNA, mRNA, or any othertranscription product, whereby either said hnRNA, mRNA or any othertranscription product, respectively, or the nucleic acid according tothe present invention is subject to a translation process, preferably acellular translation process, resulting in a biologically active RTP801Lprotein. It is to be acknowledged that a functional nucleic acid aspreferably used herein is capable of reducing the expression of a targetnucleic acid. More preferably, such reduction is based on apost-transcriptional gene silencing process of the target nucleic acid.Even more preferably such reduction is based on RNA interference. A mostpreferred form of the functional nucleic acid is an siRNA molecule orany further molecule having the same effect as an siRNA molecule. Suchfurther molecule is selected from the group comprising siRNAs, syntheticsiRNAs, shRNAs and synthetic shRNAs. As used herein siRNAs mayadditionally comprise expression vector derived siRNAs, whereby theexpression vector is in a preferred embodiment a virus such asAdenoviruses, Adenoassociated viruses, Herpes viruses and Lentiviruses.As used herein shRNA preferably means short hairpin RNAs. Such shRNA canbe made synthetically or can be generated using vector encodedexpression systems, preferably using RNA polymerase III promoters. Inconnection therewith it is to be acknowledged that the functionalnucleic acid according to the present invention is directed to RTP801Lwhich is also preferably referred to herein as the target and thenucleic acid coding for said target as the target nucleic acid.

As preferably used herein, the double-stranded structure of the nucleicacid according to the present invention comprises any double-strandedstructure, whereby such double-stranded structure is preferablygenerated by the first stretch and the second stretch provided by thenucleic acid having the basic design. The double-stranded structure maycomprise one or several mismatches. Such double-stranded structure isformed by Watson-Crick-base pairing and/or Hoogsteen base pairing and/orsimilar base pairing mechanisms. Based on the basic design of thenucleic acid according to the present invention it is preferred that onestretch, is in antisense orientation to a nucleic acid sequence codingfor RTP801L or a part thereof, whereas the other stretch is in the senseorientation to a nucleic acid sequence coding for RTP801L or a partthereof. Because of this, one stretch is complementary to a nucleic acidsequence coding for RTP801L or a part thereof, and the other stretch isidentical to a nucleic acid sequence coding for RTP801L or a partthereof. In connection therewith it is to be acknowledged that the termidentical, of course, means also partially identical, whereby theidentity, expressed as homology, is at least 80%, preferably 90%, morepreferably 95%, 96%, 97%, 98%, 99% or 100%. Similar to the definition ofidentity, complementarity can be defined in terms of homology, wherebysuch homology is of the same range as the identity if the complementarystrand would be translated into the identical strand according toWatson-Crick base pairing rules. In an alternative embodiment, onestretch is identical to a nucleic acid sequence coding for RTP801L or apart thereof and the other stretch is complementary to a nucleic acidsequence coding for RTP801L or a part thereof.

In a preferred embodiment, the nucleic acid according to the presentinvention is down-regulating RTP801L function. Down-regulation ofRTP801L function preferably happens by reduction in the level ofexpression at the protein level and/or the mRNA level, whereby suchreduced level of expression, preferably at the protein level, can be aslittle as 5% and be as high as 100%, with reference to an expressionunder conditions where the nucleic acid according to the presentinvention is not administered or is not functionally active. Suchconditions are preferably the conditions of or as present in anexpression system, preferably an expression system for RTP801L. Suchexpression system is preferably a translation system which can be an invitro translation system, more preferably a cell, organ and/or organism.It is more preferred that the organism is a multicellular organism, morepreferably a mammal, whereby such mammal is preferably selected from thegroup comprising man, monkey, mouse, rat, guinea pig, rabbit, cat, dog,sheep, cow, horse, cattle and pig. In connection with thedown-regulation it is to be acknowledged that said down-regulation maybe a function of time, i. e. the down-regulation effect is notnecessarily observed immediately upon administration or functionalactivation of the nucleic acids according to the present invention, butmay be deferred in time as well as in space, i. e. in various cells,tissues and/or organs. Such deferment may range from 5%-100%, preferably10 to 50%. It will be acknowledged by the ones skilled in the art that a5% reduction for a longer time period might be as effective as a 100%reduction over a shorter time period. It will also be acknowledged bythe ones skilled in the art that such deferment strongly depends on theparticular functional nucleic acid actually used, as well as on thetarget cell population and thus, ultimately, on the disease to betreated and/or prevented according to the technical teaching of thepresent application. Insofar, a 5% reduction over a longer time periodmight be as effective as 100% reduction over a shorter time period. Itwill also be acknowledged by the ones skilled in the art that thedeferment can occur at any level as outlined above, i.e. a deferment infunction, whereby such function is any function exhibited by RTP801L, adeferment in protein expression or a deferment at mRNA expression level.

In a preferred embodiment the first stretch comprises at least 14nucleotides, preferably 14 contiguous nucleotides. It will beacknowledged by the one skilled in the art that the first stretch shouldhave a length which is suitable to allow for specifically addressing anucleic acid sequence coding for RTP801L and more specifically thenucleic acid coding for RTP801L as present in the translation systemwhere the expression of RTP801L is to be reduced. Again without wishingto be bound by any theory or any mode of action of the nucleic acidaccording to the present invention, it seems that there is aninteraction between the nucleic acid according to the present inventionand the nucleic acid sequence coding for RTP801L, preferably at thetranscript level, i. e. upon generation of an mRNA from the respectivenucleic acid sequence coding for RTP801L. Due to the likelihood of anysequence of the nucleic acid according to the present invention beingidentical to or complementary to a sequence contained in the genome ortranscriptome of the translation system, the length of the first stretchshould thus be as long as to make sure that, under the assumption thatsome kind of base pairing between the nucleic acid coding for RTP801Land one of the strands of the nucleic acid according to the presentinvention actually occurs, only the sequence coding for RTP801L but noother coding sequence, preferably no other essential coding sequence, ofthe genome or the transcriptome is addressed for or by such basepairing. By this length, the occurrence of off-target effects can bereduced and preferably eliminated. To increase the stringency of thiskind of specifically addressing RTP801L and the nucleic acid sequencecoding therefor, the first stretch preferably has a length of at least18 or 19 nucleotides. The upper limit for the length of the firststretch is preferably less than 50 nucleotides, however, the length canbe significantly longer and can comprise 100, 200 or even 500nucleotides or any length in-between. Apart from this, one skilled inthe art will prefer to have a rather short first stretch, particularlyin case the nucleic acid according to the present invention ischemically synthesised as the shorter the sequence is, the less time andmaterial consuming the synthesis thereof will be and the lower will bethe rate at which incorrect nucleotides are inserted into the respectivesequence. Another factor which is to be taken into consideration inconnection with fixing the length of the first stretch is the fact that,typically at a length beyond 50 or more nucleotides, an unspecificinterferon response may be observed. It depends on the particularcondition to be treated whether this kind of unspecific interferonresponse is to be tolerated or not. For example, an interferon responsecould be tolerated if the interferon response and/or the expression ofthe interferon genes can be limited to the pathogenic cells.

In view of this, more preferred lengths of the first stretch are fromabout 14 to 40 nucleotides, 18 to 30 nucleotides, 19 to 27 nucleotides,21 to 25 nucleotides and 19 to 23 nucleotides.

The same considerations as outlined above for the first stretch areapplicable to the second stretch which may thus comprise any length asdescribed herein in connection with the first stretch. It is also withinthe present invention that the length of the first stretch is differentfrom the length of the second stretch, however, it is preferred thatboth stretches have the same length.

According to the basic design of the nucleic acid, the first stretch andsecond stretch are parts of the first strand and second strand,respectively, of the nucleic acid according to the present invention. Itwill be acknowledged that at either end, i. e. at the 5′ end as well asthe 3′ end the first strand and/or second strand may comprise one orseveral nucleotides, preferably additional nucleotides, at anycombination.

In connection therewith it is to be acknowledged that those nucleotidesof the individual strand going beyond the end(s) of the stretchcorresponding to the respective strand can be used to further contributeto the complementarity and identity, respectively, of the stretch andthus to the specific addressing of the nucleic acid sequence coding forRTP801L.

It will be acknowledged that, basically, based on the technical teachingprovided herein, the nucleic acid according to the present invention canaddress any part of the nucleic acid sequence coding for RTP801L,preferably coding for RTP801L in the translation system where theexpression of RTP801L is to be reduced. Insofar, the present inventioncomprises any nucleic acid having the characteristics as defined herein,whereby the complementary and identical strands and stretches of thenucleic acid according to the present invention can basically start fromany nucleotide of the nucleic acid sequence coding for RTP801L.Accordingly, under the proviso that the first stretch of the nucleicacid according to the present invention is complementary to the nucleicacid sequence coding for RTP801L, i. e. is the antisense strand thereofor is in antisense orientation thereto, the first nucleotide of saidstretch, i. e. the most 5′ terminal nucleotide corresponds, i. e. alignsto the last nucleotide of the sequence coding for RTP801L at the 3′ end.In a further embodiment such most 5′ terminal nucleotide corresponds tothe penultimate nucleotide of the nucleic acid coding for RTP801L and soon until the last position is reached which, given the length of theantisense stretch, still allows that the antisense strand of the nucleicacid according to the present invention is complementary to the nucleicacid sequence coding for RTP801L. Insofar, any nucleic acid according tothe present invention is within the present invention which could begenerated by scanning the nucleic acid sequence coding for RTP801Lstarting from the most 5′ terminal nucleotide thereof and laying overthe basic design of the nucleic acid according to the present inventionand realising the characteristics for such nucleic acid according to thepresent invention. The same considerations are applicable to theembodiments disclosed herein where the complementarity and identity ofthe nucleic acid according to the present invention is not only providedby the first stretch and second stretch, respectively, but suchcomplementarity and identity also involves one or more nucleotidesbeyond the first stretch and second stretch, respectively, then beingpart of the first strand and second strand, respectively.

It is to be noted that those nucleic acids according to the presentinvention which can be used in both human and an animal model such asrat and/or mouse and/or chinchilla are particularly useful. Thesurprising advantage of these particular nucleic acids according to thepresent invention resides in the fact that they are effective both inhuman and in an animal model which means that the test results obtainedin the animal model can be immediately transferred from the animal modelto the human being and more particularly without the necessity to makeany changes to the human sequence which would otherwise become necessaryin case the nucleic acid according to the present invention was designedsuch as to comprise (a) sequence(s) which differ(s) between the species,more particularly the species used for animal model testing and man asthe ultimate preferred organisms or patient. It is further preferredthat these nucleic acids have a modification pattern as also describedin the examples.

However, it is also within the present invention that any of thesequences according to SEQ. ID. NOs. 3-1852 and respective combinationsresulting in the nucleic acid molecules according to the presentinvention only partially contained in a further nucleic acid accordingto the present invention. Preferably, the further nucleic acidsaccording to the present invention comprise at least 14 contiguousnucleotides of the SEQ. ID. NO.s 3-1852, and more preferably 14contiguous nucleotide base pairs at any end of the double-strandedstructure comprised of the first stretch and second stretch as outlinedin the preceding table. It will be understood by the ones skilled in theart that given the potential length of the nucleic acid according to thepresent invention and particularly of the individual stretches formingsuch nucleic acid according to the present invention, some shiftsrelative to the coding sequence of RTP801L to each side is possible,whereby such shifts can be up to 1, 2, 3, 4, 5 and 6 nucleotides in bothdirections, and whereby the thus generated double-stranded nucleic acidmolecules shall also be within the present invention.

In a preferred embodiment of the present invention the first stretch andthe first strand have the same length. Likewise it is preferred that thesecond strand has the same length as the second stretch, whereby it iseven more preferred that the first stretch and the second stretch havethe same length. In a still more preferred embodiment, the first strandonly comprises the first stretch and the second strand only comprisesthe second stretch. In an even more preferred embodiment neither thefirst stretch, and thus the first strand, nor the second stretch, andthus the second strand, comprise an overhang. In other words, it is alsowithin the present invention that the double-stranded nucleic acidsaccording to the present invention are blunt ended, preferably at eachend of the double-stranded structure of the nucleic acids according tothe present invention. Such blunt ended structure can be realized inconnection with any other embodiments of the nucleic acids according tothe present invention, particularly those embodiments where the nucleicacids according to the present invention have a modification pattern,more preferably a modification pattern as described herein.

In a further aspect, the nucleic acid according to the present inventionhas thus a basic design which provides for blunt ends at both ends ofthe double-stranded structure of the nucleic acid according to thepresent invention. However, it is also within the present invention thatthere is a overhang, i. e. a stretch of one or more nucleotidesprotruding from the double-stranded structure. The overhang can be, inprinciple, at the 5′ end of the antisense strand, at the 3′ end of theantisense strand, at the 5′ end of the sense strand and/or the 3′ end ofthe sense strand. It is to be noted that realising any single of saidoptions as well as any combination thereof is within the presentinvention. More preferred is a combination, whereby the overhang islocated at the 3′ end of the antisense strand and at the 3′ end of thesense strand. It is also within the present invention that the overhangis at the 5′ end of the antisense strand and at the 5′ end of the sensestrand. Furthermore it is within the present invention that the overhangis located only at the antisense strand of double-stranded structure,more preferably at the 3′ end of the antisense strand of thedouble-stranded structure.

In connection with the overhangs, it is to be noted that the overhangplus the stretch preferably form the strand and the lengths provided forthe stretches herein apply also to these embodiments. The individualoverhang can, independent of its location, consist of at least onenucleotide. However, the individual overhang can comprise as many as 10and is preferably two nucleotides long. It is within the presentinvention that the respective nucleotide(s) forming the overhang(s)is/are also complementary to the nucleic acid sequence coding forRTP801L in case of the first strand being complementary to said nucleicacid sequence coding for RTP801L, and the overhang being at the 3′ or 5′end of the antisense strand, or that the overhang(s) is/are identical tothe nucleic acid sequence coding for RTP801L in case the first strand isidentical to the nucleic acid sequence coding for RTP801L. The sameapplies to any overhang located at the second stretch of the basicdesign of the nucleic acid according to the present invention, wherebyit is to be acknowledged that the overhang design at the second stretchcan be independent from the overhang design of the first stretch.

It is also within the present invention that the overhang formingnucleotides are neither complementary nor identical to the correspondingnucleotides of the nucleic acid sequence coding for RTP801L. As usedherein, and preferably in this embodiment, “corresponding” means therespective nucleotides which follow at the 5′ end and/or the 3′ end ofthe stretch having a nucleotide counterpart on the nucleic acid codingfor RTP801L.

Preferably, the first strand comprises at its 3′ end two nucleotides,preferably deoxynucleotides and more preferably two TT and/or this kindof nucleotides also at the 3′ end of the second strand, whereby morepreferably the length of the first stretch and the second stretch is 19nucleotides. The strands are thus comprised of the stretch and theoverhang. In this embodiment the double-stranded structure consists of19 base pairs and an overhang of two nucleotides at each end of 3′ endof the individual stretch.

In a preferred embodiment, the first stretch and/or the first strandcomprise(s) ribonucleotides, whereby it is particularly preferred thatthe first stretch consists in its entirety of ribonucleotides. The sameapplies to the second stretch and the second strand, respectively. Inconnection therewith, however, each and any of the nucleotides of thefirst stretch and second stretch, respectively, is modified in apreferred embodiment. The same applies to the first strand and secondstrand, respectively. Particularly the terminal nucleotides,irrespective whether they are ribonucleotides or deoxyribonucleotides,can have an OH-group which as such can be modified. Such OH-group maystem from either the sugar moiety of the nucleotide, more preferablyfrom the 5′position in case of the 5′OH-group and/or from the 3′positionin case of the 3′OH-group, or from a phosphate group attached to thesugar moiety of the respective terminal nucleotide. The phosphate groupmay in principle be attached to any OH-group of the sugar moiety of thenucleotide. Preferably, the phosphate group is attached to the5′OH-group of the sugar moiety in case of the free 5′OH-group and/or tothe 3′OH-group of the sugar moiety in case of the free 3′OH-group stillproviding what is referred to herein as free 5′ or 3′ OH-group.

As used herein with any strategy for the design of RNAi or anyembodiment of RNAi disclosed herein, the term end modification means achemical entity added to the most 5′ or 3′ nucleotide of the firstand/or second strand. Examples for such end modifications include, butare not limited to, 3′ or 5′ phosphate, inverted (deoxy) abasics, amino,fluoro, chloro, bromo, CN, CF, methoxy, imidazole, caboxylate, thioate,C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl,OCF₃, OCN, O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂, N₃; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;polyalkylamino or substituted silyl, as, among others, described inEuropean patents EP 0 586 520 B1 or EP 0 618 925 B1.

As used herein, alkyl or any term comprising “alkyl” preferably meansany carbon atom chain comprising 1 to 12, preferably 1 to 6 and more,preferably 1 to 2 C atoms.

A further end modification is a biotin group. Such biotin group maypreferably be attached to either the most 5′ or the most 3′ nucleotideof the first and/or second strand or to both ends. In a more preferredembodiment the biotin group is coupled to a polypeptide or a protein. Itis also within the scope of the present invention that the polypeptideor protein is attached through any of the other aforementioned endmodifications. The polypeptide or protein may confer furthercharacteristics to the nucleic acid molecules according to the presentinvention. Among others the polypeptide or protein may act as a ligandto another molecule. If said other molecule is a receptor the receptor'sfunction and activity may be activated by the binding ligand. Thereceptor may show an internalization activity which allows an effectivetransfection of the ligand bound nucleic acid molecules according to thepresent invention. An example for the ligand to be coupled to theinventive nucleic acid molecule is VEGF and the corresponding receptoris the VEGF receptor.

Various possible embodiments of the RNAi of the present invention havingdifferent kinds of end modification(s) are presented in the followingTable 1.

TABLE 1 VARIOUS EMBODIMENTS OF THE INTERFERING RIBONUCLEIC ACIDACCORDING TO THE PRESENT INVENTION 1^(st) strand/1^(st) stretch 2^(nd)strand/2nd stretch 1.) 5′-end free OH free OH 3′-end free OH free OH 2.)5′-end free OH free OH 3′-end end modification end modification 3.)5′-end free OH free OH 3′-end free OH end modification 4.) 5′-end freeOH free OH 3′-end end modification free OH 5.) 5′-end free OH endmodification 3′-end free OH free OH 6.) 5′-end free OH end modification3′-end end modification free OH 7.) 5′-end free OH end modification3′-end free OH end modification 8.) 5′-end free OH end modification3′-end end modification end modification

The various end modifications as disclosed herein are preferably locatedat the ribose moiety of a nucleotide of the nucleic acid according tothe present invention. More particularly, the end modification may beattached to or replace any of the OH-groups of the ribose moiety,including but not limited to the 2′OH, 3′OH and 5′OH position, providedthat the nucleotide thus modified is a terminal nucleotide. Invertedabasics are nucleotides, either desoxyribonucleotides or ribonucleotideswhich do not have a nucleobase moiety. This kind of compound is, amongothers, described in Sternberger, M., Schmiedeknecht, A., Kretschmer,A., Gebhardt, F., Leenders, F., Czauderna, F., Von Carlowitz, I., Engle,M., Giese, K., Beigelman, L. & Klippel, A. (2002). Antisense NucleicAcid Drug Dev, 12, 131-43

Any of the aforementioned end modifications may be used in connectionwith the various embodiments of RNAi depicted in Table 1; it is to benoted that the 5′ end modifications mentioned above are usually onlypresent in the sense strand of the siRNA molecule

Further modifications can be related to the nucleobase moiety, the sugarmoiety or the phosphate moiety of the individual nucleotide.

Such modification of the nucleobase moiety can be such that thederivatives of adenine, guanine, cytosine and thymidine and uracil,respectively, are modified. Particularly preferred modified nucleobasesare selected from the group comprising inosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl, 2-propyl and other alkyladenines, 5-halouracil, 5-halocytosine, 5-halo cytosine, 6-azacytosine, 6-aza thymine,pseudo-uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thioladenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiolguanine, 8-thioalkyl guanine, 8-hydroxylguanine and other substitutedguanines, other aza- and deaza adenines, other aza- and deaza guanines,5-trifluoromethyl uracil and 5-trifluoro cytosine.

In another preferred embodiment, the sugar moiety of the nucleotide ismodified, whereby such modification preferably is at the 2′ position ofthe ribose and desoxyribose moiety, respectively, of the nucleotide.More preferably, the 2′ OH group is replaced by a group or moietyselected from the group comprising amino, fluoro, alkoxy and alkyl.Preferably, alkoxy is either methoxy or ethoxy. Also preferably alkylmeans methyl, ethyl, propyl, isobutyl, butyl and isobutyl. It is evenmore preferred that, regardless of the type of modification, thenucleotide is preferably a ribonucleotide.

The modification of the phosphate moiety is preferably selected from thegroup comprising phosphothioates.

It will be acknowledged by the one skilled in the art that the nucleicacid of the present invention which consists of a multitude ofnucleotides may thus be formed by nucleotides which are linked through aphosphodiester linkage or through a phosphothioate linkage, or acombination of both along the length of the nucleotide sequence of theindividual strand and stretch, respectively.

A further form of nucleotides used may be siNA which is, among others,described in international patent application WO 03/070918.

The nucleotides forming the first stretch and first strand,respectively, of the nucleic acid according to the present invention cancomprise one or more modified nucleotides, whereby the individualmodified nucleotide has a modification which is preferably amodification as disclosed herein. In addition to the particularmodification, the modification can be or comprise some sort of label,whereby the label is selected from the group chemiluminescent labels,fluorescent labels and radio labels. These kinds of labels are known tothe one skilled in the art and, e. g., described in Ausubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,Md., 1998. The thus labelled nucleic acid according to the presentinvention may be used also for diagnostic purposes or for monitoring thesite of action as well as for the staging of any treatment, preferablyrelated to any of the diseases disclosed herein.

In a preferred embodiment, the nucleic acid according to the presentinvention is modified such that the pyrimidine nucleotides in the sensestretch or strand are 2′ O-methyl pyrimidine nucleotides and, eitheradditionally or alternatively, the purine nucleotides in the sensestretch or strand are 2′-deoxypurine nucleotides. In a furtherembodiment the pyrimidine nucleotides present in the sense stretch orsense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides.

In an alternative embodiment, the modification is not based on thechemistry of the nucleotide, i. e. the modification depends on whetherthe nucleotide to be modified is either a purine nucleotide or apyrimidine nucleotide, but is predominantly based on the individualnucleotide's spatial arrangement in the overall double-strandedstructure of the basic design of the nucleic acid according to thepresent invention.

More particularly, either the first strand and first stretch,respectively, or the second strand and second stretch, respectively,show a spatial pattern of modification of the nucleotides forming saidstretches and strands, respectively.

Focusing on the first stretch first, there is a pattern of groups ofmodified nucleotides and groups of non-modified nucleotides. Thesegroups of non-modified nucleotides are also referred to herein asflanking groups of nucleotides. More preferably, the pattern consists ofgroups of modified nucleotides and non-modified nucleotides. Even morepreferably, the pattern is a regular pattern and even more preferably analternating pattern along the length of the first stretch of the nucleicacid according to the present invention. The group of modifiednucleotides may either consist of one or of several nucleotides whichare modified and which are preferably nucleotides which are modified atthe 2′ position, i. e. have a modification at the sugar moiety. Morepreferably, this modification is a 2′-O-Me modification.

The group of non-modified nucleotides may either consist of one or ofseveral nucleotides which are either not modified, whereby thenot-modified nucleotides are preferably ribonucleotides, or the notmodified nucleotides are nucleotides having a modification, whereby suchmodification is different from the modification shown by the nucleotidesforming the group of modified nucleotides. Even more preferably, the notmodified nucleotides are ribonucleotides. It is to be noted that theterm not modified and non-modified nucleotide are used in aninterchangeable manner if not indicated to the contrary. The firststretch of the nucleic acid according to the present invention mayeither start with a group of modified nucleotides or start with a groupof non-modified nucleotides as defined herein. However, it is preferredthat the first stretch starts with a group of modified nucleotides. Mostpreferably, the group of modified nucleotides consists of a singlenucleotide. In connection with this embodiment the first stretch ispreferably in antisense orientation to the nucleic acid coding forRTP801L. It is also within the present invention that the modificationas exhibited by the nucleotides forming the group of modifiednucleotides is the same for all groups of modified nucleotides presenton the first stretch. However, it is also within the present inventionthat some group of modified nucleotides have a different modificationthan one or several groups of modified nucleotides present on the firststretch.

On the second strand of the nucleic acid according to the presentinvention, a pattern as described for the first stretch can also berealised. The same characteristics as described in connection with thefirst stretch can be realized in an embodiment on the second stretch aswell, whereby it is preferred that, under the proviso that the secondstretch is in sense orientation relative to the nucleic acid sequencecoding for RTP801L, the second strand of the nucleic acid according tothe present invention starts with a group of non-modified nucleotides.

The nucleic acid according to the present invention comprising adouble-stranded structure may comprise a first stretch having themodification pattern as described herein. Alternatively, thedouble-stranded nucleic acid according to the present invention maycomprise a second stretch having the modification pattern as outlinedabove. It is, however, most preferred that the double-stranded nucleicacid according to the present invention consists of a first stretch anda second stretch, whereby both the first stretch and the second stretchhave a spatial modification pattern as described herein.

It is within the present invention that the characteristics of thespatial modification pattern is the same on both stretches in terms ofsize of the groups of modified nucleotides and groups of non-modifiednucleotides and the kind of modifications actually used. Preferably, thespatial pattern of modification on the first stretch is shifted suchthat a group of modified nucleotides on the first stretch is opposing agroup of non-modified nucleotides on the second stretch and vice versa.However, it is also with the present invention that the patterns areexactly aligned, i. e. that a group of modified nucleotides on the firststretch is opposing a group of non-modified nucleotides on the secondstretch and a group of non-modified nucleotides on the first stretch isopposing a group of non-modified nucleotides on the second stretch. Itis still within the present invention that the spatial pattern ofmodification on the first stretch and the second stretch is shiftedrelative to each other so that only a first portion of a group ofmodified nucleotides on one stretch is opposing a portion of a group ofnon-modified nucleotides on the other stretch, whereas the secondportion of the group of modified nucleotides is opposing another groupof modified nucleotides. It is within the present invention that thedisclosure provided herein on the spatial modification pattern of thestretch(es) of the nucleic acid according to the present inventionapplies also to the strand(s) of the nucleic acid according to thepresent invention. However, it is preferred that the stretches of thenucleic acid comprise the spatial modification pattern and the strandscomprise such stretches and one or more overhang(s) as disclosed herein.It is particularly preferred that there is a phosphate group at the 3′end of either the antisense strand, or the sense strand or both strands,whereby it is more preferred that the phosphate group is at the 3′ endof both the antisense strand and the sense strand. In an even morepreferred embodiment, the phosphate group is a phosphate group asdefined herein.

It is also within the present invention that the nucleic acid accordingto the present invention may exhibit a linker connecting the first andthe second strand. Such linker is preferably a polymer. The polymer canbe any synthetic or natural polymer. Possible synthetic linkers are,among others, PEG or a polynucleotide. Such linker is preferablydesigned such as to allow the either partial or complete folding back ofthe first stretch onto the second stretch and vice versa.

Finally, it is within the present invention that the nucleic acidaccording to the present invention is a synthetic one, a chemicallysynthesised one, an isolated one, or one derived from any naturalsources such as, for example, prepared by means of recombinanttechnology. In connection with the preparation of any nucleic acidaccording to the present invention any modification as disclosed hereincan be introduced either prior, during or subsequent to the preparationof the respective nucleic acid according to the present invention asknown to the ones skilled in the art.

The vector according to the present invention comprises a nucleic acidaccording to the present invention. Additionally, the vector may includeelements to control targeting, expression and transcription of saidnucleic acid in a cell selective manner as is known in the art. Theplasmid can include a promoter for controlling transcription of theheterologous material, i. e. the nucleic acid according to the presentinvention, and can be either a constitutive or an inducible promoter toallow selective transcription. Enhancers that may be required to obtainnecessary transcription levels can optionally be included. Enhancers aregenerally any non-translated DNA sequences which work contiguously withthe coding sequence, thus in cis, to change the basal transcriptionlevel dictated by the promoter. The expression of such constructs isknown to the one skilled in the art and may be done, e. g., by providinga respective tandem construct or by having different promoterstranscribing for the first and second strand and first and secondstretch, respectively, of the nucleic acid according to the presentinvention.

When the nucleic acid according to the present invention is manufacturedor expressed, preferably expressed in vivo, more preferably in a patientwho is in need of the nucleic acid according to the present invention,such manufacture or expression preferably uses an expression vector,preferably a mammalian expression vector. Expression vectors are knownin the art and preferably comprise plasmids, cosmids, viral expressionsystems. Preferred viral expression systems include, but are not limitedto, adenovirus, retrovirus and lentivirus.

Methods are known in the art to introduce the vectors into cells ortissues. Such methods can be found generally described in Sambrook etal., Molecular cloning: A Laboratory Manual, Cold Springs HarbourLaboratory, New York (1983, 1992), or in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.,1998.

Suitable methods comprise, among others, transfection, lipofection,electroporation and infection with recombinant viral vectors. Inconnection with the present invention, an additional feature of thevector is in one embodiment an expression limiting feature such as apromoter and regulatory element, respectively, that are specific for thedesired cell type thus allowing the expression of the nucleic acidsequence according to the present invention only once the background isprovided which allows the desired expression.

In a further aspect the present invention is related to a pharmaceuticalcomposition comprising a nucleic acid according to the present inventionand/or a vector according to the present invention and, optionally, apharmaceutically acceptable carrier, diluent or adjuvants or othervehicle(s). Preferably, such carrier, diluents, adjuvants and vehiclesare inert, and non-toxic. The pharmaceutical composition is in itsvarious embodiments adapted for administration in various ways. Suchadministration comprises systemic and local administration as well asoral, subcutaneous, parenteral, intravenous, intraarterial,intramuscular, intraperitonial, intranasal, and intrategral.

It will be acknowledged by the ones skilled in the art that the amountof the pharmaceutical composition and the respective nucleic acid andvector, respectively, depends on the clinical condition of theindividual patient, the site and method of administration, scheduling ofadministration, patient age, sex, bodyweight and other factors known tomedical practitioners. The pharmaceutically effective amount forpurposes of prevention and/or treatment is thus determined by suchconsiderations as are known in the medical arts. Preferably, the amountis effective to achieve improvement including but limited to improve thediseased condition or to provide for a more rapid recovery, improvementor elimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the medical arts.

In a preferred embodiment, the pharmaceutical composition according tothe present invention may comprise other pharmaceutically activecompounds. Preferably, such other pharmaceutically active compounds areselected from the group comprising compounds which allow for uptakeintracellular cell delivery, compounds which allow for endosomalrelease, compounds which allow for, longer circulation time andcompounds which allow for targeting of endothelial cells or pathogeniccells. Preferred compounds for endosomal release are chloroquine, andinhibitors of ATP dependent H⁺ pumps.

The pharmaceutical composition is preferably formulated so as to providefor a single dosage administration or a multi-dosage administration.

It will be acknowledged that the pharmaceutical composition according tothe present invention can be used for any disease which involvesundesired development or growth of vasculature including angiogenesis,as well as any of the diseases and conditions described herein.Preferably, these kind of diseases are tumor diseases. Among tumordiseases, the following tumors are most preferred: endometrial cancer,colorectal carcinomas, gliomas, endometrial cancers, adenocarcinomas,endometrial hyperplasias, Cowden's syndrome, hereditary non-polyposiscolorectal carcinoma, Li-Fraumene's syndrome, breast-ovarian cancer,prostate cancer (Ali, I. U., Journal of the National Cancer Institute,Vol. 92, no. 11, Jun. 7, 2000, page 861-863), Bannayan-Zonana syndrome,LDD (Lhermitte-Duklos' syndrome) (Macleod, K., supra)hamartoma-macrocephaly diseases including Cow disease (CD) andBannayan-Ruvalcaba-Rily syndrome (BRR), mucocutaneous lesions (e. g.trichilemmonmas), macrocephaly, mental retardation, gastrointestinalharmatomas, lipomas, thyroid adenomas, fibrocystic disease of thebreast, cerebellar dysplastic gangliocytoma and breast and thyroidmalignancies (Vazquez, F., Sellers, W. R., supra).

It is to be acknowledged that any of the tumor disease to be treatedwith the pharmaceutical composition according to the present inventionis preferably a late stage tumor disease. In another embodiment, thetumor disease involves cells which are tumor suppressor negative,whereby more preferably the tumor suppressor is PTEN.

The pharmaceutical composition according to the present invention canalso be used in a method for preventing and/or treating a disease asdisclosed herein, whereby the method comprises the administration of anucleic acid according to the present invention, a vector according tothe present invention or a pharmaceutical composition or medicamentaccording to the present invention for any of the diseases describedherein.

In a further aspect, the present invention is related to a method fordesigning or screening a nucleic acid which is suitable to down-regulateRTP801L, more particularly to down-regulate RTP801L function. Thismethod comprises the use of a nucleic acid sequence as disclosed hereinand the assessment of such nucleic acid in a suitable assay. Such assayis known in the art and, for example, described in the example part ofthis application. In a further step, a double-stranded nucleic acid isdesigned, preferably according to the design principles as laid downherein, which is suitable to down-regulate RTP801L, preferably inconnection with a post transcriptional gene silencing mechanism such asRNA interference. Also the thus obtained, i. e. designed or screened,nucleic acid is assessed in the respective assay and the result, i. e.the effect of both the nucleic acid according to the present inventionas well as the newly designed or screened nucleic acid in such assaycompared. Preferably, the designed or screened nucleic acid is moresuitable in case it is either more stable or more effective, preferablyboth. It will be acknowledged that the method will be particularlyeffective if any of the nucleic acids according to the present inventionis used as a starting point. It is thus within the present inventionthat new nucleic acid molecules will be designed based on the principlesdisclosed herein, whereby the target sequence on the RTP801L mRNA willbe slightly shifted relative to the target sequence on the RTP801L mRNAfor the corresponding nucleic acid according to the present invention.Preferably the new nucleic acid will be shifted by at least one or morenucleotides relative to the stretch on the target mRNA in either the 5′or the 3′ direction of the mRNA coding for RTP801L. It is however within the present invention that the shift occurs in both directionssimultaneously which means that the new nucleic acid incorporates thenucleic acid according to the present invention used as a startingpoint. It is also within the present invention that the elongation ofthe nucleic acid according to the present invention and used as astarting point is biased to either the 3′ end or the 5′ end. In case ofsuch as bias either the 3′ end or the 5′ end of the new nucleic acid islonger, i.e more extended than the other end. When the new nucleic acidmolecule is generated by extending either the 3′ end of the 5′ end ofthe antisense strand and/or the sense strand, the following sequence ofsteps is typically applied. If the shift is to the 5′ end of the mRNA ofRTP801L, the 3′ end of the antisense strand has to be extended by thenumber of the nucleotides by which the 5′ end of the mRNA of RTP801L isshifted. The nucleotide(s) thus to be added to the 3′ end of theantisense strand of the new nucleic acid is/are complementary to thosenucleotides following at the 5′ end of the target sequence on theRTP801L mRNA used for the nucleic acid molecule according to the presentinvention used as a starting point. The same has to be done to the sensestrand. However the nucleotides to be added to the sense strand have tocorrespond, i.e. be complementary to the nucleotides newly added to the3′ end of the antisense strand which means that they have to be added tothe 5′ end of the sense strand. The latter step on the sense strand,however has to be done only to the extent that apart from the antisensestrand also the sense strand shall be shifted, which is the case inpreferred embodiments of the present invention. Although this shiftingcan be done to an extent defined by the ones skilled in the art, morepreferably the shift shall be done such that also the new nucleic acidstill contains a strech of at least 14 nucleotides, preferably 14contiguous nucleotides as exhibited by any of the nucleic acid moleculesdisclosed herein.

The synthesis of any of the nucleic acids described herein is within theskills of the one of the art. Such synthesis is, among others, describedin Beaucage S. L. and Iyer R. P., Tetrahedron 1992; 48: 2223-2311,Beaucage S. L. and Iyer R. P., Tetrahedron 1993; 49: 6123-6194 andCaruthers M. H. et. al., Methods Enzymol. 1987; 154: 287-313, thesynthesis of thioates is, among others, described in Eckstein F., Annu.Rev. Biochem. 1985; 54: 367-402, the synthesis of RNA molecules isdescribed in Sproat B., in Humana Press 2005 Edited by Herdewijn P.;Kap. 2: 17-31 and respective downstream processes are, among others,described in Pingoud A. et. al., in IRL Press 1989 Edited by Oliver R.W. A.; Kap. 7: 183-208 and Sproat B., in Humana Press 2005 Edited byHerdewijn P.; Kap. 2: 17-31 (supra).

siRNA for RTP801L can be made using methods known in the art asdescribed above, based on the known sequence of RTP801L (SEQ ID NO:1),and can be made stable by various modifications as described above. Forfurther information, see Example 5.

Further, in relation to the methods of the present invention asdescribed herein, additional RNA molecules may be used with said methodse.g. inhibitory RNA molecules of the present invention include singlestranded oligoribonucleotides preferably comprising stretches of atleast 7-10 consecutive nucleotides present in the sequences detailed inTable A, said oligoribonucleotides being capable of forming [and/orcomprising] double stranded regions in particular conformations that arerecognized by intracellular complexes, leading to the degradation ofsaid oligoribonucleotides into smaller RNA molecules that are capable ofexerting inhibition of their corresponding endogenous gene, and DNAmolecules encoding such RNA molecules. The corresponding endogenous geneis preferably the 801L gene and may additionally be the RTP801 gene(described in U.S. Pat. No. 6,555,667), the VEGF gene and/or theVEGF-R1gene. The invention also provides a composition comprising theabove single stranded oligoribonucleotide in a carrier, preferably apharmaceutically acceptable carrier.

Additionally, the present invention provides for combination therapy forall the conditions disclosed herein and in particular conditionsinvolving choroidal neovascularization. In said combination therapy,both the RTP801L and VEGFR genes are inhibited in order to amelioratethe symptoms of the disease being treated. These genes may be inhibitedwith a combination of siRNAs or antibodies (including aptamerantibodies) or both. The present invention therefore also provides for anovel pharmaceutical composition comprising an RTP801L inhibitor and aVEGF or VEGFR-1 inhibitor, the RTP801L inhibitor preferable being ansiRNA, more preferably an siRNA molecule detailed in Table A, optionally-selected from the group consisting of siRNAs Nos: 72 and 73, and theVEGF/ VEGFR-1 inhibitor optionally being an antibody or aptamer. Thecombined use of said compounds (i.e., RTP801L siRNA and VEGF antibody orany other combined example disclosed herein) in the preparation of amedicament is also part of the present invention.

Thus, RTP801L siRNA such as an siRNA molecule detailed in Table A andoptionally siRNA Nos: 72 and 73, may be administered in conjunction withagents which target VEGF or VEGF receptor 1 (VEGFR1). Such agentscurrently exist on the market or in various stages of approval and workthrough different mechanisms. Antibodies and antibody fragments such asranibizumab (Lucentis, Genentech) attach to released VEGF to inhibitbinding of VEGF to active receptors. An aptamer which can act like aligand/antibody (Macugen, Eyetech/Pfizer, approved recently by the FDAfor wet AMD) is also a possibility. Macugen bonds with extracellularVEGF to block its activity. These drugs are administered locally byintravitreal injection. Anti-VEGF siRNA based compounds (such asAcuity's Cand5 inhibitor of VEGF or SIRNA's 027 inhibitor of VEGFR-1)are also available. Additionally, the small molecule aminosterolSqualamine (Genaera) which is administered systemically reportedlyinterferes in multiple facets of the angiogenic process, includinginhibiting VEGF and other growth factor signaling in endothelial cells.

The conjoined administration of an RTP801L inhibitor, preferably ansiRNA, and any of the above VEGF/VEGFR-1 inhibitory agents can have anadditive or even synergistic effect whereby said combined treatment ismore effective than treatment by any of these individual compositions,irrespective of dosage in the single therapy option. RTP801L siRNA has adifferent mechanism of action and is potentially additive or evensynergistic with VEGF-VEGFR inhibitors.

It is to be understood that, in the context of the present invention,any of the siRNA molecules disclosed herein, or any long double-strandedRNA molecules (typically 25-500 nucleotides in length) which areprocessed by endogenous cellular complexes (such as DICER—see above) toform the siRNA molecules disclosed herein, or molecules which comprisethe siRNA molecules disclosed herein, can be employed in the treatmentof the diseases or disorders described herein.

Additional disorders which can be treated by the molecules andcompositions of the present invention include all types of choroidalneovascularization (CNV), which occurs not only in wet AMD but also inother ocular pathologies such as ocular histoplasmosis syndrome, angiodstreaks, ruptures in Bruch's membrane, myopic degeneration, oculartumors and some retinal degenerative diseases.

An additional aspect of the present invention provides for methods oftreating an apoptosis related disease. Methods for therapy of diseasesor disorders associated with uncontrolled, pathological cell growth,e.g. cancer, psoriasis, autoimmune diseases, inter alia, and methods fortherapy of diseases associated with ischemia and lack of proper bloodflow, e.g. myocardial infarction (MI) and stroke, are provided. “Cancer”or “Tumor” refers to an uncontrolled growing mass of abnormal cells.These terms include both primary tumors, which may be benign ormalignant, as well as secondary tumors, or metastases which have spreadto other sites in the body. Examples of cancer-type diseases include,inter alia: carcinoma (e.g.: breast, colon and lung), leukemia such as Bcell leukemia, lymphoma such as B-cell lymphoma, blastoma such asneuroblastoma and melanoma and sarcoma.

The invention also provides a composition comprising one or more of thecompounds of the invention in a carrier, preferably a pharmaceuticallyacceptable carrier. This composition may comprise a mixture of two ormore siRNAs for different genes or different siRNAs for the same gene. Acomposition comprising siRNA for the RTP801L gene and siRNA for the VEGFgene and/or the VEGF-R1 gene is envisaged.

Another compound of the invention comprises the above compound of theinvention (structure A) covalently or non-covalently bound to one ormore compounds of the invention (structure A). This compound may bedelivered in a carrier, preferably a pharmaceutically acceptablecarrier, and may be processed intracellularly by endogenous cellularcomplexes to produce one or more siRNAs of the invention. Anothercompound of the invention comprises the above compound of the invention(structure A) covalently or non-covalently bound to an siRNA for anothergene, especially the VEGF gene and/or the VEGF-R1 gene.

This invention also comprises a novel chemical entity which is anRTP801L inhibitor, preferably an siRNA, chemically bound, covalently ornon-covalently, to any of the above VEGF/VEGFR-1 inhibitory agents. Aparticular chemical entity envisaged is an siRNA RTP801L inhibitorcovalently bound to an antibody to VEGF or VEGF receptor-1. Methods ofproduction of such novel chemical entities are known to those skilled inthe art.

This invention also comprises a tandem double-stranded structure whichcomprises two or more siRNA sequences, which is processedintracellularly to form two or more different siRNAs, one inhibiting 801and a second inhibiting VEGF/VEGFR-1 In a related aspect, this inventionalso comprises a tandem double-stranded structure which comprises two ormore siRNA sequences, which is degraded intracellularly to form two ormore different siRNAs, both inhibiting 801.

In particular, it is envisaged that a long oligonucleotide (typicallyabout 80-500 nucleotides in length) comprising one or more stem and loopstructures, where stem regions comprise the sequences of theoligonucleotides of the invention, may be delivered in a carrier,preferably a pharmaceutically acceptable carrier, and may be processedintracellularly by endogenous cellular complexes (e.g. by DROSHA andDICER as described above) to produce one or more smaller double strandedoligonucleotides (siRNAs) which are oligonucleotides of the invention.This oligonucleotide can be termed a tandem shRNA construct. It isenvisaged that this long oligonucleotide is a single strandedoligonucleotide comprising one or more stem and loop structures, whereineach stem region comprises a sense and corresponding antisense siRNAsequence of an 801 gene. In particular, it is envisaged that thisoligonucleotide comprises sense and antisense siRNA sequences asdepicted in Table A. Alternatively, the tandem shRNA construct maycomprise sense and complementary antisense siRNA sequence correspondingto an 801L gene and additionally sense and complementary antisense siRNAsequence corresponding to a different gene such as 801, VEGF or VEGF-R1.

As mentioned herein, siRNA against RTP801L may be the main activecomponent in a pharmaceutical composition, or may be one activecomponent of a pharmaceutical composition containing two or more siRNAs(or molecules which encode or endogenously produce two or more siRNAs,be it a mixture of molecules or one or more tandem molecule whichencodes two or more siRNAs), said pharmaceutical composition furtherbeing comprised of one or more additional siRNA molecule which targetsone or more additional gene. Simultaneous inhibition of RTP801L and saidadditional gene(s) has an additive or synergistic effect for treatmentof the diseases disclosed herein, according to the following:

Acute Renal Failure (ARF) and other microvascular disorders: thepharmaceutical composition for treatment of ARF may be comprised of thefollowing compound combinations: 1) RTP801L siRNA and p53 siRNA dimers;2) RTP801L and Fas siRNA dimers; 3) RTP801L and Bax siRNA dimers; 4) p53and Fas siRNA dimers; 5) RTP801L and Bax siRNA dimers; 6) RTP801L andNoxa siRNA dimers; 7) RTP801L and Puma siRNA dimers; 8) RTP801L (REDD1)and RTP801LL (REDD2) siRNA dimers; 9) RTP801L siRNA, Fas siRNA and anyof RTP801LL siRNA p53 siRNA, Bax siRNA, Noxa siRNA or Puma siRNA to formtrimers or polymers (i.e., tandem molecules which encode three siRNAs).

Macular degeneration (MD), diabetic retinopathy (DR), spinal cordinjury: pharmaceutical compositions for treatment of MD, DR and spinalcord injury may be comprised of the following compound combinations: 1)RTP801L siRNA combined with either of VEGF siRNA, VEGF-R1 siRNA, VEGF R2siRNA, PKCbeta siRNA, MCP1 siRNA, eNOS siRNA, KLF2 siRNA, RTP801 siRNA(either physically mixed or in a tandem molecule); 2) RTP801L siRNA incombination with two or more siRNAs of the above list (physically mixedor in a tandem molecule encoding three siRNAs, or a combinationthereof).

COPD and respiratory disorders: the pharmaceutical composition fortreatment of respiratory disorders may be comprised of the followingcompound combinations: RTP801L siRNA combined with siRNA against one ormore of the following genes: elastases, matrix metalloproteases,phospholipases, caspases, sphingomyelinase, RTP801 and ceramidesynthase.

Further, a combination (tandem) siRNA directed against both RTP801 andRTP801L can be used to treat any of the conditions disclosed herein. ForExample, the siRNA directed against RTP801 termed REDD14 (sensesequence: 5′ GUGCCAACCUGAUGCAGCU 3′ and antisense sequence 5′AGCUGCAUCAGGUUGGCAC 3′) can be joined in tandem with any of the RTP801LsiRNAs disclosed herein, such as siRNA No.s 72 or 73 in Table A, or anyother siRNA present in Table A

Additionally, RTP801L siRNA or any nucleic acid molecule comprising orencoding RTP801L siRNA can be linked (covalently or non-covalently) toantibodies, in order to achieve enhanced targeting for treatment of thediseases disclosed herein, according to the following:

ARF: anti-Fas antibody (preferably neutralizing antibodies).

Macular degeneration, diabetic retinopathy, spinal cord injury: anti-Fasantibody, anti-MCP1 antibody, anti-VEGFR1 and anti-VEGFR2 antibody. Theantibodies should be preferably be neutralizing antibodies.

Any molecules, such as, for example, antisense DNA molecules whichcomprise the siRNA sequences disclosed herein (with the appropriatenucleic acid modifications) are particularly desirable and may be usedin the same capacity as their corresponding siRNAs for all uses andmethods disclosed herein.

The invention also comprises a method of treating a patient sufferingfrom a disorder such as the disorders described herein comprisingadministering to the patient the above composition or compound in atherapeutically effective dose so as to thereby treat the patient.

Antisense Molecules

By the term “antisense” (AS) or “antisense fragment” is meant apolynucleotide fragment (comprising either deoxyribonucleotides,ribonucleotides or a mixture of both) having inhibitory antisenseactivity, said activity causing a decrease in the expression of theendogenous genomic copy of the corresponding gene. An AS polynucleotideis a polynucleotide which comprises consecutive nucleotides having asequence of sufficient length and homology to a sequence present withinthe sequence of the target gene to permit hybridization of the AS to thegene. Many reviews have covered the main aspects of antisense (AS)technology and its enormous therapeutic potential (Aboul-Fadl T., CurrMed Chem. 2005; 12(19):2193-214; Crooke S T, Curr Mol Med. 2004 August;4(5):465-87; Crooke S T, Annu Rev Med. 2004; 55:61-95; Vacek M et al.,Cell Mol Life Sci. 2003 May; 60(5):825-33; Cho-Chung Y S, Arch PharmRes. 2003 March; 26(3):183-91. There are further reviews on the chemical(Crooke, 1995; Uhlmann et al, 1990), cellular (Wagner, 1994) andtherapeutic (Hanania, et al, 1995; Scanlon, et al, 1995; Gewirtz, 1993)aspects of this technology. Antisense intervention in the expression ofspecific genes can be achieved by the use of synthetic ASoligonucleotide sequences (for recent reports see Lefebvre-d'Hellencourtet al, 1995; Agrawal, 1996; LevLehman et al, 1997).

AS oligonucleotide sequences may be short sequences of DNA, typically15-30 mer but may be as small as 7 mer (Wagner et al, 1996), designed tocomplement a target mRNA of interest and form an RNA:AS duplex. Thisduplex formation can prevent processing, splicing, transport ortranslation of the relevant mRNA. Moreover, certain AS nucleotidesequences can elicit cellular RNase H activity when hybridized withtheir target mRNA, resulting in mRNA degradation (Calabretta et al, 1996Semin Oncol. 23(1):78-87). In that case, RNase H will cleave the RNAcomponent of the duplex and can potentially release the AS to furtherhybridize with additional molecules of the target RNA. An additionalmode of action results from the interaction of AS with genomic DNA toform a triple helix which can be transcriptionally inactive.

The sequence target segment for the antisense oligonucleotide isselected such that the sequence exhibits suitable energy relatedcharacteristics important for oligonucleotide duplex formation withtheir complementary templates, and shows a low potential forself-dimerization or self-complementation [Anazodo et al., 19961. Forexample, the computer program OLIGO (Primer Analysis Software, Version3.4), can be used to determine antisense sequence melting temperature,free energy properties, and to estimate potential self-dimer formationand self-complimentary properties. The program allows the determinationof a qualitative estimation of these two parameters (potentialself-dimer formation and self- complimentary) and provides an indicationof “no potential” or “some potential” or “essentially completepotential”. Using this program target segments are generally selectedthat have estimates of no potential in these parameters. However,segments can be used that have “some potential” in one of thecategories. A balance of the parameters is used in the selection as isknown in the art. Further, the oligonucleotides are also selected asneeded so that analogue substitution do not substantially affectfunction.

Phosphorothioate antisense oligonucleotides do not normally showsignificant toxicity at concentrations that are effective and exhibitsufficient pharmacodynamic half-lives in animals (Agarwal et al., 1996)and are nuclease resistant. Antisense induced loss-of-functionphenotypes related with cellular development were shown for the glialfibrillary acidic protein (GFAP), for the establishment of tectal plateformation in chick (Galileo et al., 1991) and for the N-myc protein,responsible for the maintenance of cellular heterogeneity inneuroectodermal cultures (ephithelial vs. neuroblastic cells, whichdiffer in their colony forming abilities, tumorigenicity and adherence)(Rosolen et al., 1990; Whitesell et al, 1991). Antisense oligonucleotideinhibition of basic fibroblast growth factor (bFgF), having mitogenicand angiogenic properties, suppressed 80% of growth in glioma cells(Morrison, 1991) in a saturable and specific manner. Being hydrophobic,antisense oligonucleotides interact well with phospholipid membranes(Akhter et al., 1991). Following their interaction with the cellularplasma membrane, they are actively (or passively) transported intoliving cells (Loke et al., 1989), in a saturable mechanism predicted toinvolve specific receptors (Yakubov et al., 1989).

Ribozymes

A “ribozyme” is an RNA molecule that possesses RNA catalytic ability(see Cech for review) and cleaves a specific site in a target RNA. Inaccordance with the present invention, ribozymes which cleave mRNA maybe utilized as inhibitors. This may be necessary in cases whereantisense therapy is limited by stoichiometric considerations (Sarver etal., 1990, Gene Regulation and Aids, pp. 305-325). Ribozymes can then beused that will target the a gene associated with a bone marrow disease.The number of RNA molecules that are cleaved by a ribozyme is greaterthan the number predicted by stochiochemistry. (Hampel and Tritz, 1989;Uhlenbeck, 1987).

Ribozymes catalyze the phosphodiester bond cleavage of RNA. Severalribozyme structural families have been identified including Group Iintrons, RNase P, the hepatitis delta virus ribozyme, hammerheadribozymes and the hairpin ribozyme originally derived from the negativestrand of the tobacco ringspot virus satellite RNA (sTRSV) (Sullivan,1994; U.S. Pat. No. 5,225,347). The latter two families are derived fromviroids, and virusoids, in which the ribozyme is believed to separatemonomers from oligomers created during rolling circle replication(Symons, 1989 and 1992). Hammerhead and hairpin ribozyme motifs are mostcommonly adapted for trans-cleavage of mRNAs for gene therapy (Sullivan,1994). In general the ribozyme has a length of from about 30-100nucleotides. Delivery of ribozymes is similar to that of AS fragmentsand/or siRNA molecules.

It will be noted that all the polynucleotides to be used in the presentinvention may undergo modifications so as to possess improvedtherapeutic properties. Modifications or analogs of nucleotides can beintroduced to improve the therapeutic properties of polynucleotides.Improved properties include increased nuclease resistance and/orincreased ability to permeate cell membranes. Nuclease resistance, whereneeded, is provided by any method known in the art that does notinterfere with biological activity of the AS polynucleotide, siRNA, cDNAand/or ribozymes as needed for the method of use and delivery (Iyer etal., 1990; Eckstein, 1985; Spitzer and Eckstein, 1988; Woolf et al.,1990; Shaw et al., 1991). Modifications that can be made tooligonucleotides in order to enhance nuclease resistance includemodifying the phophorous or oxygen heteroatom in the phosphate backbone.These include preparing methyl phosphonates, phosphorothioates,phosphorodithioates and morpholino oligomers. In one embodiment it isprovided by having phosphorothioate bonds linking between the four tosix 3′-terminus nucleotide bases. Alternatively, phosphorothioate bondslink all the nucleotide bases. Other modifications known in the art maybe used where the biological activity is retained, but the stability tonucleases is substantially increased.

All analogues of, or modifications to, a polynucleotide may be employedwith the present invention, provided that said analogue or modificationdoes not substantially affect the function of the polynucleotide. Thenucleotides can be selected from naturally occurring or syntheticmodified bases. Naturally occurring bases include adenine, guanine,cytosine, thymine and uracil. Modified bases of nucleotides includeinosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl, 2-propyl andother alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and6-aza thymine, psuedo uracil, 4-thiuracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine andother substituted guanines, other aza and deaza adenines, other aza anddeaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

In addition, analogues of polynucleotides can be prepared wherein thestructure of the nucleotide is fundamentally altered and that are bettersuited as therapeutic or experimental reagents. An example of anucleotide analogue is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA is replacedwith a polyamide backbone which is similar to that found in peptides.PNA analogues have been shown to be resistant to degradation by enzymesand to have extended lives in vivo and in vitro. Further, PNAs have beenshown to bind stronger to a complementary DNA sequence than a DNAmolecule. This observation is attributed to the lack of charge repulsionbetween the PNA strand and the DNA strand. Other modifications that canbe made to oligonucleotides include polymer backbones, cyclic backbones,or acyclic backbones, as well as LNA (“locked nucleic acid”).

The polypeptides employed in the present invention may also be modified,optionally chemically modified, in order to improve their therapeuticactivity. “Chemically modified”—when referring to the polypeptides,means a polypeptide where at least one of its amino acid residues ismodified either by natural processes, such as processing or otherpost-translational modifications, or by chemical modification techniqueswhich are well known in the art. Among the numerous known modificationstypical, but not exclusive examples include: acetylation, acylation,amidation, ADP-ribosylation, glycosylation, GPI anchor formation,covalent attachment of a lipid or lipid derivative, methylation,myristlyation, pegylation, prenylation, phosphorylation, ubiqutination,or any similar process.

Additional possible polypeptide modifications (such as those resultingfrom nucleic acid sequence alteration) include the following:

“Conservative substitution”—refers to the substitution of an amino acidin one class by an amino acid of the same class, where a class isdefined by common physicochemical amino acid side chain properties andhigh substitution frequencies in homologous polypeptides found innature, as determined, for example, by a standard Dayhoff frequencyexchange matrix or BLOSUM matrix. Six general classes of amino acid sidechains have been categorized and include: Class I (Cys); Class II (Ser,Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg,Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). Forexample, substitution of an Asp for another class III residue such asAsn, Gln, or Glu, is a conservative substitution.

“Non-conservative substitution”—refers to the substitution of an aminoacid in one class with an amino acid from another class; for example,substitution of an Ala, a class II residue, with a class III residuesuch as Asp, Asn, Glu, or Gln.

“Deletion”—is a change in either nucleotide or amino acid sequence inwhich one or more nucleotides or amino acid residues, respectively, areabsent.

“Insertion” or “addition”—is that change in a nucleotide or amino acidsequence which has resulted in the addition of one or more nucleotidesor amino acid residues, respectively, as compared to the naturallyoccurring sequence.

“Substitution”—replacement of one or more nucleotides or amino acids bydifferent nucleotides or amino acids, respectively. As regards aminoacid sequences the substitution may be conservative or non-conservative.

In an additional embodiment of the present invention, the RTP801Lpolypeptide or polynucleotide may be used to diagnose or detect maculardegeneration in a subject. A detection method would typically compriseassaying for RTP801L mRNA or RTP801L polypeptide in a sample derivedfrom a subject.

“Detection”—refers to a method of detection of a disease. This term mayrefer to detection of a predisposition to a disease, or to the detectionof the severity of the disease.

By “homolog/homology”, as utilized in the present invention, is meant atleast about 70%, preferably at least about 75% homology, advantageouslyat least about 80% homology, more advantageously at least about 90%homology, even more advantageously at least about 95%, e.g., at leastabout 97%, about 98%, about 99% or even about 100% homology. Theinvention also comprehends that these polynucleotides and polypeptidescan be used in the same fashion as the herein or aforementionedpolynucleotides and polypeptides.

Alternatively or additionally, “homology”, with respect to sequences,can refer to the number of positions with identical nucleotides or aminoacid residues, divided by the number of nucleotides or amino acidresidues in the shorter of the two sequences, wherein alignment of thetwo sequences can be determined in accordance with the Wilbur and Lipmanalgorithm ((1983) Proc. Natl. Acad. Sci. USA 80:726); for instance,using a window size of 20 nucleotides, a word length of 4 nucleotides,and a gap penalty of 4, computer-assisted analysis and interpretation ofthe sequence data, including alignment, can be conveniently performedusing commercially available programs (e.g., Intelligenetics™ Suite,Intelligenetics Inc., CA). When RNA sequences are said to be similar, orto have a degree of sequence identity or homology with DNA sequences,thymidine (T) in the DNA sequence is considered equal to uracil (U) inthe RNA sequence. RNA sequences within the scope of the invention can bederived from DNA sequences or their complements, by substitutingthymidine (T) in the DNA sequence with uracil (U).

Additionally or alternatively, amino acid sequence similarity orhomology can be determined, for instance, using the BlastP program(Altschul et al., Nucl. Acids Res. 25:3389-3402) and available at NCBI.The following references provide algorithms for comparing the relativeidentity or homology of amino acid residues of two polypeptides, andadditionally, or alternatively, with respect to the foregoing, theteachings in these references can be used for determining percenthomology: Smith et al., (1981) Adv. Appl. Math. 2:482-489; Smith et al.,(1983) Nucl. Acids Res. 11:2205-2220; Devereux et al., (1984) Nucl.Acids Res. 12:387-395; Feng et al., (1987) J. Molec. Evol. 25:351-360;Higgins et al., (1989) CABIOS 5:151-153; and Thompson et al., (1994)Nucl. Acids Res. 22:4673-4680.

“Having at least X % homolgy”—with respect to two amino acid ornucleotide sequences, refers to the percentage of residues that areidentical in the two sequences when the sequences are optimally aligned.Thus, 90% amino acid sequence identity means that 90% of the amino acidsin two or more optimally aligned polypeptide sequences are identical.

An additional embodiment of the present invention concerns apharmaceutical composition comprising an RTP801L inhibitor in atherapeutically affective amount as an active ingredient and apharmaceutically acceptable carrier. The inhibitor may be a biologicalinhibitor, an organic molecule, a chemical molecule, etc. saidpharmaceutical composition may comprise an RTP801L inhibitor which is apolynucleotide which comprises consecutive nucleotides having a sequencewhich is an antisense sequence to the sequence set forth in FIG. 1 (SEQID No: 1). Further, the RTP801L inhibitor may be a vector comprisingthese polynucleotides. Additionally, the RTP801L inhibitor may be amonoclonal antibody which specifically binds to an epitope comprising4-25 amino acids set forth in FIG. 2 (SEQ ID No:2), or an RNA moleculewhich targets the RTP801L gene mRNA such as an siRNA molecule(optionally depicted in Table A) or a ribozyme.

The active ingredients of the pharmaceutical composition can includeoligonucleotides that are nuclease resistant needed for the practice ofthe invention or a fragment thereof shown to have the same effecttargeted against the appropriate sequence(s) and/or ribozymes.Combinations of active ingredients as disclosed in the present inventioncan be used, including combinations of antisense sequences.

An additional embodiment of the present invention provides for the useof a therapeutically effective dose of an RTP801L inhibitor for thepreparation of a medicament for promoting recovery in a patientsuffering from any of the diseases or conditions described herein egspinal cord disease or injury. In one embodiment the inhibitor ispreferably an siRNA. In another embodiment the inhibitor is preferablyStructure A depicted herein.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventioncan be practiced otherwise than as specifically described.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year and patents by number.The disclosures of these publications and patents and patentapplications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart to which this invention pertains.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Standard molecular biology protocols known in the art not specificallydescribed herein are generally followed essentially as in Sambrook etal., Molecular cloning: A laboratory manual, Cold Springs HarborLaboratory, New-York (1989, 1992), and in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1988).

Standard organic synthesis protocols known in the art not specificallydescribed herein are generally followed essentially as in Organicsyntheses: Vol. 1- 79, editors vary, J.

Wiley, New York, (1941-2003); Gewert et al., Organic synthesis workbook,Wiley-VCH, Weinheim (2000); Smith & March, Advanced Organic Chemistry,Wiley-Interscience; 5th edition (2001).

Standard medicinal chemistry methods known in the art not specificallydescribed herein are generally followed essentially as in the series“Comprehensive Medicinal Chemistry”, by various authors and editors,published by Pergamon Press.

The features of the present invention disclosed in the specification,the claims and/or the drawings may both separately and in anycombination thereof be material for realizing the invention in variousforms thereof.

Example 1

General Materials and Methods

If not indicated to the contrary, the following materials and methodswere used in Examples 1-5:

Cell Culture

The first human cell line, namely HeLa cells (American Type CultureCollection) were cultured as follows: Hela cells (American Type CultureCollection) were cultured as described in Czauderna F et al. (Czauderna,F., Fechtner, M., Aygun, H., Arnold, W., Klippel, A., Giese, K. &Kaufmann, J. (2003). Nucleic Acids Res, 31, 670-82).

The second human cell line was a human keratinozyte cell line which wascultivated as follows: Human keratinocytes were cultured at 37° C. inDulbecco's modified Eagle medium (DMEM) containing 10% FCS.

The mouse cell line was B16V (American Type Culture Collection) culturedat 37° C. in Dulbecco's modified Eagle medium (DMEM) containing 10% FCS.Culture conditions were as described in Methods Find Exp Clin Pharmacol.1997 May; 19(4):231-9:

In each case, the cells were subject to the experiments as describedherein at a density of about 50,000 cells per well and thedouble-stranded nucleic acid according to the present invention wasadded at 20 nM, whereby the double-stranded nucleic acid was complexedusing 1 μg/ml of a proprietary lipid.

Induction of Hypoxia-like Condition

The cells were treated with CoCl₂ for inducing a hypoxia-like conditionas follows: siRNA transfections were carried out in 10-cm plates (30-50%confluency) as described by (Czauderna et al., 2003; Kretschmer et al.,2003). Briefly, siRNA were transfected by adding a preformed 10×concentrated complex of GB and lipid in serum-free medium to cells incomplete medium. The total transfection volume was 10 ml. The finallipid concentration was 1.0 μg/ml; the final siRNA concentration was 20nM unless otherwise stated. Induction of the hypoxic responses wascarried out by adding CoCl₂ (100 μM) directly to the tissue culturemedium 24 h before lysis.

Preparation of Cell Extracts and Immuno Blotting

The preparation of cell extracts and immuno blot analysis were carriedout essentially as described by Klippel et al. (Klippel, A., Escobedo,M. A., Wachowicz, M. S., Apell, G., Brown, T. W., Giedlin, M. A.,Kavanaugh, W. M. & Williams, L. T. (1998). Mol Cell Biol, 18, 5699-711;Klippel, A., Reinhard, C., Kavanaugh, W. M., Apell, G., Escobedo, M. A.& Williams, L. T. (1996). Mol Cell Biol, 16, 4117-27). Polyclonalantibodies against full length RTP801L were generated by immunisingrabbits with recombinant RTP801L protein producing bacteria from pET19-bexpression vector (Merck Biosciences GmbH, Schwalbach, Germany). Themurine monoclonal anti-p110a and anti-p85 antibodies have been describedby Klippel et al. (supra).

Example 2

Experimental Models, Methods and Results Relating to MacularDegeneration

The compounds of the present invention are tested in the followinganimal model of Choroidal neovascularization (CNV). This hallmark of wetAMD is induced in model animals by laser treatment.

A) Mouse Model

Choroidal Neovascularization (CNV) Induction

Choroid neovascularization (CNV), a hallmark of wet AMD, is triggered bylaser photocoagulation (532 nm, 200 mW, 100 ms, 75 μm) (OcuLight G L,Iridex, Mountain View, Calif.) performed on both eyes of each mouse onday 0 by a single individual masked to drug group assignment. Laserspots are applied in a standardized fashion around the optic nerve,using a slit lamp delivery system and a cover slip as a contact lens.

Treatment Groups

CNV is induced in the following groups of mice (males 6-8 weeks of age):

-   -   (1) 12 WT mice;    -   (2) 12 RTP801L Knock-Out mice;    -   (3) 12 WT mice injected with 0.25 μg of synthetic stabilized        active anti-RTP801L siRNA (REDD14) in one eye and inactive        anti-RTP801L siRNA (REDD8-negative control) in the fellow eye,        at days 0 and 7;    -   (4) 12 WT mice injected with 0.25 μg of synthetic stabilized        active anti-RTP801L siRNA (REDD14) in one eye and inactive        anti-GFP siRNA (negative control) in the fellow eye at days 0        and 7;    -   (5) 12 WT mice injected with either 0.1 μg of synthetic        stabilized active anti-RTP801L siRNA (REDD14) in one eye and PBS        (negative control) in the fellow eye at days 0 and 7;    -   (6) 12 WT mice injected with either 0.05 μg of synthetic        stabilized active anti-RTP801L siRNA (REDD14) in one eye and PBS        (negative control) in the fellow eye at days 0 and 7.

Both eyes of each mouse are laser-treated.

Evaluation

-   -   1. The experiment is terminated at day 14. For evaluation, the        eyes are enucleated and fixed with 4% paraformaldehyde for 30        min at 4° C. The neurosensory retina is detached and severed        from the optic nerve. The remaining RPE-choroid-sclera complex        is flat mounted in Immu-Mount (Vectashield Mounting Medium,        Vector) and coverslipped. Flat mounts are examined with a        scanning laser confocal microscope (TCS SP, Leica, Germany).        Vessels are visualized by exciting with blue argon laser.        Horizontal optical sections (1 μm step) are obtained from the        surface of the RPE-choroid-sclera complex. The deepest focal        plane in which the surrounding choroidal vascular network        connecting to the lesion can be identified is judged to be the        floor of the lesion. Any vessel in the laser treated area and        superficial to this reference plane is judged as CNV. Images of        each section are digitally stored. The area of CNV-related        fluorescence is measured by computerized image analysis using        the Leica TCS SP software. The summation of whole fluorescent        area in each horizontal section is used as an index for the        volume of CNV.    -   2. Separate WT mice are used for evaluating RTP801L mRNA        expression in CNV (as well as the expression of other genes        relevant to AMD) (untreated and treated with siRNA) using        real-time PCR on RNA extracted from RPE/choroids, or from neural        retina.

Expression profiling conducted in the mouse model of CNV revealed thatthe RTP801L transcript level is gradually increased in mouse Retinafollowing CNV induction, thus indicating that RTP801L is a good targetfor inhibition in the treatment of AMD and other conditions whichinvolve choroidal neovascularization.

B) Non-Human Primate Model

CNV Induction

Choroidal neovascularization (CNV) is induced by perimacular lasertreatment of both eyes prior to doseadministration. Nine lesions areplaced in the macula with a laser [OcuLight GL (532 nm) LaserPhoto-coagulator with an IRIS Medical® Portable Slit Lamp Adaptor], andlaser spots in the right eye mirror the placement in the left eye. Theapproximate laser parameters are as follows: spot size: 50-100 μmdiameter; laser power: 300-700 milliwatts; exposure time: 0.1 seconds.

Treatment

Immediately following laser treatment, both eyes of all animals aresubjected to a single intravitreal injection. Left eye is typicalitydosed with 350 ug of synthetic stabilized siRNA against RTP801L in thefinal volume of 50 ul, whereas the contralateral eye receives 50 ul ofPBS (vehicle).

Evaluation

-   -   1. All the animals are subjected to daily examination of food        consumption and body weight measurements.    -   2. two monkeys are euthanized at day 6 following CNV induction.        Their eyes are enucleated and the posterior pole is flattened.        Then the fovea region is excised and separated into choroids and        neuroretina which are separately (for every animal) frozen in        liquid nitrogen to be subsequentlyused for RNA extraction and        real time PCR evaluation of RTP801L expression.    -   3. Fluorescein angiograms are performed pre-study, and at the        end of weeks 1, 2, and 3 following CNV induction. Photographs        are taken, using a fundus camera (TRC-50EX Retina Camera).        Images are captured using the TOPCON IMAGEnet™ system.        Fluorescein dye (10% fluorescein sodium, approximately 0.1        mL/kg) is injected via vascular access ports. Photographs are        taken at several timepoints following dye injection, to include        the arterial phase, early arteriovenous phase and several late        arteriovenous phases in order to evaluate neovascularization snd        to monitor leakage of fluorescein associated with CNV lesions.        Interpretation and analysis of the fluorescein angiograms is        independently conducted by two ophthalmologists.

Neovascularization (NV) is assessed in early angiograms and every spotis graded according to the following scheme:

-   -   0—no signs of NV    -   0.5—suspicious spot    -   1—“hot” spot    -   2—NV in the laser burn    -   3—evident NV

Leakage is assessed according to the following scheme:

-   -   0—no leakage    -   0.5—suspicious spot    -   1—evident small spot leakage    -   2—leakage growing with time    -   3—leakage greater than previous borders (evidently)

In addition, the size of every spot is compared between the early andthe late angiograms using morphometric measurements, and the increase inthe spot's size resulting from the leakage is calculated.

-   -   4. Electroretinograms (ERGs) are recorded using an Epic 2000        electroretinograph according to Sierra's SOPs and the        study-specific SOP, including the use of the Ganzfield        apparatus, at prestudy and in the end of week 3. The tabulated        ERG data are evaluated by a veterinary ophthalmologist.

C) Efficacy of Combination Therapy of RTP801L siRNA (REDD14) andAnti-VEGF Antibody

The efficacy of combination therapy of RTP801L siRNA (REDD14) andanti-VEGF antibody or aptamer (such as macugen) in the treatment ofdiseases in which CNV occurs is tested in the above mouse CNV model.

A) CNV Volume Studies

The volume of choroidal neovascularization (CNV) 3 weeks after laserinjury is computed byconfocal fluorescence microscopy as previouslydescribed (Sakurai et al. IOVS 2003; 44: 3578-85 & Sakurai et al. IOVS2003; 44: 2743-2749).

In previous studies the assignee of the present invention found thatanti-VEGF-A antibody (Ab) reduced CNV volume in a dose dependentfashion. A dose of 1 ng of VEGF-A Ab was chosen for the RTP801LsiRNA+VEGF-A Ab combination studies because this dose had anintermediate inhibitory effect: VEGF-A Ab (1 ng) reduced the size of CNVby 26±6%.

In a stusy conducted with siRNA against RTP801, The principal findingswere:

-   -   The addition of RTP801 siRNA at the lower 0.05 μg dose reduced        the size of CNV by 27±4% compared to VEGF-A Ab alone.    -   The addition of RTP801 siRNA at the higher 0.25 μg dose reduced        the size of CNV by 55±3% compared to VEGF-A Ab alone.

B) CNV Leakage Studies

Experiment 1

This experiment was designed in order to identify a potential additiveor synergistic therapeutic effect of inhibition of VEGF and RTP801L inthe model of laser-induced choroid neovascularization in mice

Materials:

-   -   RTP801L siRNA    -   negative control    -   Anti-VEGF antibodies or macugen    -   negative control

CNV is induced on day zero as described above; the test material isinjected to the subjects on day zero and day 7.

The results are evaluated by Fluorescein angiography on weeks 1, 2, 3,and by CNV volume measurement on week 3.

Experimental Groups:

-   -   VEGF Ab or macugen 0.5 ng/eye    -   VEGF Ab or macugen 1 ng/eye    -   VEGF Ab or macugen 2 ng/eye    -   VEGF Ab 4 or macugen ng/eye    -   RTP801L siRNA 0.05 ug/eye    -   RTP801L siRNA 0.1 ug/eye    -   RTP801L siRNA 0.25 ug/eye    -   RTP801L siRNA 0.05 ug/eye+VEGF Ab or macugen 1 ng/eye    -   RTP801L siRNA 0.1 ug/eye+VEGF Ab or macugen 1 ng/eye    -   RTP801L siRNA 0.25 ug/eye+VEGF Ab or macugen 1 ng/eye

Control Groups

-   -   PBS    -   Non-specific IgG 2 ng/eye    -   negative control 0.1 ug/eye    -   negative control 0.1 ug/eye+VEGF Ab or macugen 1 ng/eye

The results show an additive or synergistic therapeutic effect ofinhibition of VEGF and RTP801L

Experiment 2

This experiment was designed in order to study the effect of RTP801LsiRNA on gene expression in RPE and neural retina.

Experimental Design

Groups:

-   -   PBS    -   RTP801L siRNA 0.25 mg

CNV is induced by laser treatment as described above on day zero; thetest material is also injected on day zero, and the effect evaluated byqPCR analysis of gene expression in RPE and neural retina on days zeroand 5.

Results Obtained with RTP801 a Functionally Homologous Gene:

-   -   Simultaneous inhibition of RTP801L and VEGF has enhanced        inhibitory effect on choroid neovascularization and neovascular        leakage.    -   Inhibition of RTP801 expression by REDD14 (siRNA to 801) not        only prevents PEDF downregulation in the CNV model but enhances        its expression compared to the baseline.    -   Inhibition of RTP801 expression leads to concomitant        downregulation of MCP1 which should have an anti-inflammatory        effect.    -   Without being bound by theory, the increase of PEDF expression        by REDD14 may underlie the observed cooperative effect of        simultaneous inhibition of VEGF and RTP801    -   (Note: PEDF is a well-known antiangiogenic and neuroprotective        factor.)    -   Without being bound by theory, the reduction of MCP1 expression        by REDD 14 may also underlie the observed cooperative effect of        simultaneous inhibition of VEGF and RTP801    -   (Note: MCP1 is a known pro-inflammatory chemokine involved in        pathogenesis of AMD.)

Similar results are obtained using inhibitors to 801L.

Additional AMD models which may be used to test the methods of thepresent invention:

-   -   Ccl-2 or Ccr-2 deficient animals—deficiency in either of these        proteins causes the development of some of the main features of        AMD. Animals deficient in these proteins can be used to test the        methods of the present invention.

For further information on AMD animal models, see: Chader, Visionresearch 42 (2002) 393-399; Ambati et al., Nature Medicine 9(11) (2003)1390-1397; Tolentino et al., Retina 24 (2004) 132-138.

Example 3

Models and Results Relating to COPD and Emphysema

The compounds of the present invention are tested in the following ananimal models and are shown to prevent emphysema:

-   -   Cigarette smoke-induced emphysema model: chronic exposure to        cigarette smoke causes emphysema in several animals such as,        inter alia, mouse, guinea pig.    -   Lung protease activity as a trigger of emphysema.    -   VEGFR inhibition model of emphysema.    -   Bronchial instillation with human neutrophil/pancreatic elastase        in rodents.    -   MMP (matrix metalloprotease)-induced enphysema.    -   Inflammation-induced emphysema.

Additionally, emphysema models may be generated through genetic means(e.g., mice carrying the TSK mutation), and emphysematous animals may begenerated by known modifiers of susceptibility to emphysema such as,inter alia, lung injury, alveolar hypoplasia, hyperoxia, glucocorticoidtreatment and nutrition.

Evaluation of the Influence of Lack of RTP801L on Disease Progression inMouse Models of Emphysema by Inhibiting Endogenous RTP801L EmployingIntralung Delivery RTP801L -Inactivating siRNA

CS-induced inflammation is induced by 7 day smoking in 2 groups ofC57BL6 mice, 10 mice per group. Group 1: CS+delivery of control siRNA;Group 2: CS+RTP801L siRNA. Control groups of mice are instilled witheither type of siRNA but kept in room air conditions. The lungs aresubsequently agarose-inflated, fixed and imbedded in paraffin, anddevelopment oxidative stress in the KO mice is assessed by:

-   -   a) immunohistochemical localization and quantitation of 8-oxo-dG        in the lung sections;    -   b) immunohistochemical localization and quantitation of active        caspase 3 in the lung sections using specific antibodies, or        quantitative evaluation of the number of TUNEL-positive cells;    -   c) measurement of ceramide concentration in the lung extracts;    -   d) measurement of caspase activity in the lung extracts.

Methods

Exposure to Cigarette Smoking (CS)

Exposure is carried out (7 h/day, 7 days/week) by burning 2R4F referencecigarettes (2.45 mg nicotine per cigarette; purchased from the TobaccoResearch Institute, University of Kentucky, Lexington, Ky., USA) using asmoking machine (Model TE-10, Teague Enterprises, Davis, Calif., USA).Each smoldering cigarette is puffed for 2 s, once every minute for atotal of eight puffs, at a flow rate of 1.05 L/min, to provide astandard puff of 35 cm3. The smoke machine is adjusted to produce amixture of sidestream smoke (89%) and mainstream smoke (11%) by burningfive cigarettes at one time. Chamber atmosphere is monitored for totalsuspended particulates and carbon monoxide, with concentrations of 90mg/m3 and 350 ppm, respectively.

Morphologic and Morphometric Analyses

After exposing the mice to CS or instillation of RTP801L expressingplasmid, the mice are anesthetized with halothane and the lungs areinflated with 0.5% low-melting agarose at a constant pressure of 25 cmas previously described. The inflated lungs are fixed in 10% bufferedformalin and embedded in paraffin. Sections (5 μm) are stained withhematoxylin and eosin. Mean alveolar diameter, alveolar length, and meanlinear intercepts are determined by computer-assisted morphometry withthe Image Pro Plus software (Media Cybernetics, Silver Spring, Md.,USA). The lung sections in each group are coded and representativeimages (15 per lung section) are acquired by an investigator masked tothe identity of the slides, with a Nikon E800 microscope, 20× lens. Theresults show that siRNA to 801L prevents emphysema caused by smoking asmeasured by the four parameters described above.

Bronchoalveolar Lavage (BAL) and Phenotyping

Following exposure to CS or instillation of RTP801L expressing plasmid,the mice are anesthetized with sodium pentobarbital. The BAL fluidcollected from the lungs of the mice is centrifuged (500 ′g at 4° C.),and the cell pellet is resuspended in phosphate-buffered saline. Thetotal number of cells in the lavage fluid is determined, and 2×104 cellsare cytocentrifuged (Shandon Southern Products, Pittsburgh, Pa., USA)onto glass slides and stained with Wright-Giemsa stain. Differentialcell counts are performed on 300 cells, according to standard cytologictechniques.

Identification of Alveolar Apoptotic Cell Populations in the Lungs.

To identify the different alveolar cell types undergoing apoptosis inthe lungs, an immunohistochemical staining of active caspase 3 isperformed in the lung sections from the room air (RA) as well as CSexposed mice. To identify the apoptotic type II epithelial cells in thelungs, after active caspase 3 labeling, the lung sections are incubatedfirst with anti-mouse surfactant protein C (SpC) antibody and then withan anti-rabbit Texas red antibody. Apoptotic endothelial cells areidentified by incubating the sections first with the anti-mouse CD 31antibody and then with the biotinylated rabbit anti-mouse secondaryantibody. The lung sections are rinsed in PBS and then incubated withthe streptavidin-Texas red conjugated complex. The apoptotic macrophagesin the lungs are identified by incubating the sections first with therat anti-mouse Mac-3 antibody and then with the anti-rat Texas redantibody. Finally, DAPI is applied to all lung sections, incubated for 5minutes, washed and mounted with Vectashield HardSet mounting medium.DAPI and fluorescein are visualized at 330-380 nm and 465-495 nm,respectively. Images of the lung sections are acquired with the NikonE800 microscope, 40× lens.

Immunohistochemical Localization of Active Caspase-3

Immunohistochemical staining of active caspase-3 assay is performedusing anti-active caspase-3 antibody and the active caspase-3-positivecells are counted with a macro, using Image Pro Plus program. The countsare normalized by the sum of the alveolar profiles herein named asalveolar length and expressed in μm. Alveolar length correlatesinversely with mean linear intercept, i.e., as the alveolar septa aredestroyed, mean linear intercepts increases as total alveolar length,i.e., total alveolar septal length decreases.

Caspase 3 Activity Assay

The caspase-3/7 activity is measured in lung tissue extracts using afluorometric assay according to the manufacturer's instructions.Snap-frozen lung tissue (n=3 per group) was homogenized with the assaybuffer, followed by sonication and centrifugation at 800×g. Afterremoval of nuclei and cellular debris, the supernatant (300 μg protein)is then incubated with the pro-fluorescent substrate at room temperaturefor 1 h and the fluorescence intensity was measured utilizing a Typhoonphosphoimager (Amersham Biosciences, Inc., Piscataway, N.J., USA). Theresults are expressed as the rate of specific caspase-3 substratecleavage, expressed in units of caspase 3 enzymatic activity, normalizedby total protein concentration. Active recombinant caspase 3 wasutilized as the assay standard (0-4 U). Tissue lysates withoutsubstrate, assay buffer alone, and lysates with caspase 3 inhibitor wereutilized as negative controls.

Immunohistochemical Localization of 8-oxo-dG

For the immunohistochemical localization and quantification of 8-oxo-dG,lung sections from the mice exposed to CS or instilled with RTP801Lexpressing plasmid are incubated with anti-8-oxo-dG antibody and stainedusing InnoGenexTM Iso-IHC DAB kit using mouse antibodies. The8-oxo-dG-positive cells are counted with a macro (using Image Pro Plus),and the counts were normalized by alveolar length as described.

Instillation of Plasmid DNA into Mouse Lungs

Plasmid DNA of RTP801L expressing and control vectors are prepared underendotoxin-free DNA isolation kit. For intra-tracheal instillation, 50 ugof plasmid DNA is delivered in 80 ul sterile perfluorocarbon. The oxygencarrying properties of perfluorocarbon make it well-tolerated at thesevolumes, while its physical-chemical properties allow for extremelyefficient distal lung delivery when instilled intratracheally. Mice areanesthetized by brief inhalational halothane exposure, the tongue isgently pulled forward by forceps and the trachea instilled withperfluorocarbon solution applied at the base of the tongue via a bluntangiocatheter.

Instillation of siRNA into Mouse Lungs.

Mice are anesthetized with an intra-peritoneal injection ofKetamine/Xylazine (115/22 mg/kg). 50 μg of siRNA is instilledintranasally in 500 volume of 0.9% NaCl by delivering five consecutive10 μl portions. At the end of the intranasal instillation, the mouse'shead is held straight up for 1 minute to ensure that all the instilledsolution drains inside.

For further information, see: Rangasamy T, Cho C Y, Thimmulappa, R K,Then L, Srisuma S S, Kensler T W, Yamamoto M, Petrache I, Tuder R M,Biswal S. Genetic ablation of Nrf2 enhances susceptibility to cigarettesmoke-iduced emphysema in mice. Submitted to Journal of ClinincalInvestigation; Yasunori Kasahara, Rubin M. Tuder, Carlyne D. Cool, DavidA. Lynch, Sonia C. Flores, and Norbert F. Voelkel. Endothelial CellDeath and Decreased Expression of Vascular Endothelial Growth Factor andVascular Endothelial Growth Factor Receptor 2 in Emphysema. Am J RespirCrit Care Med Vol 163. pp 737-744, 2001; Yasunori Kasahara, Rubin M.Tuder, Laimute Taraseviciene-Stewart, Timothy D. Le Cras, Steven Abman,Peter K. Hirth, Johannes Waltenberger, and Norbert F. Voelkel.Inhibition of VEGF receptors causes lung cell apoptosis and emphysema.J. Clin. Invest. 106:1311-1319 (2000); and a review on the topic: RobinM. Tuder, Sharon McGrath and Enid Neptune, The pathological mechanismsof emphysema models: what do they have in common?, PulmonaryPharmacology & Therpaeutics 2002.

Example 4

Models and Results Relating to Microvascular Disorders

The compounds of the present invention are tested in animal models of arange of microvascular disorders as described below.

1. Diabetic Retinopathy

RTP801L promotes neuronal cell apoptosis and generation of reactiveoxygen species in vitro. The inventor of the current invention alsofound that in RTP801 knockout (KO) mice subjected to the model ofretinopathy of prematurity (ROP), pathologic neovascularization NV wasreduced under hypoxic conditions, despite elevations in VEGF, whereasthe lack of this gene did not influence physiologic neonatal retinal NV.Moreover, in this model, lack of RTP801 was also protective againsthypoxic neuronal apoptosis and hyperoxic vaso-obliteration.

Experiment 1

Diabetes is induced in 8 wk old RTP801L KO and C57/129sv wildtype (WT)littermate mice by intraperitoneal injection of STZ. After 4 weeks, ERG(single white flash, 1.4×10{circumflex over (0)}4 ftc, 5 ms) is obtainedfrom the left eye after 1 hour of dark adaptation. RVP is assessed fromboth eyes using the Evans-blue albumin permeation technique.

Experiment 2

Diabetes is induced in RTP801L knockout and in control wild type micewith the matched genetic background. In addition, it is induced inC57B16 mice, which are subsequently used for intravitreal injection ofanti-RTP801L and control siRNAs. For diabetes induction, the mice areinjected with streptozotocin (STZ 90 mg/kg/d for 2 days after overnightfast). Animal physiology is monitored throughout the study for changesin blood glucose, body weight, and hematocrit. Vehicle-injected miceserve as controls. The appropriate animals are treated by intravitrealinjections of 1 ug of REDD14 anti-RTP801L siRNA or lug of anti-GFPcontrol siRNA. siRNA is injected twice in the course of the study—on day0, when the first STZ injection is performed, and on day 14 after theSTZ injection.

Retinal vascular leakage is measured using the Evans-blue (EB) dyetechnique on the animals after 4 weeks duration of diabetes. Mice have acatheter implanted into the right jugular vein 24 hours prior to EvansBlue (EB) measurements. Retinal permeability measurements in both eyesof each animal follows a standard Evans-blue protocol.

2. Retinopathy of Prematurity

Retinopathy of prematurity is induced by exposing the test animals tohypoxic and hyperoxic conditions, and subsequently testing the effectson the retina.

3. Myocardial Infarction

Myocardial infarction is induced by Left Anterior Descending arteryligation in mice, both short term and long term.

4. Microvascular Ischemic Conditions

Animal models for assessing ischemic conditions include:

-   -   1. Closed Head Injury (CHI)—Experimental TBI produces a series        of events contributing to neurological and neurometabolic        cascades, which are related to the degree and extent of        behavioral deficits. CHI is induced under anesthesia, while a        weight is allowed to free-fall from a prefixed height (Chen et        al, J. Neurotrauma 13, 557, 1996) over the exposed skull        covering the left hemisphere in the midcoronal plane.    -   2. Transient middle cerebral artery occlusion (MCAO)—a 90 to 120        minutes transient focal ischemia is performed in adult, male        Sprague Dawley rats, 300-370 gr. The method employed is the        intraluminal suture MCAO (Longa et al., Stroke, 30, 84, 1989,        and Dogan et al., J. Neurochem. 72, 765, 1999). Briefly, under        halothane anesthesia, a 3-0-nylon suture material coated with        Poly-L-Lysine is inserted into the right internal carotid artery        (ICA) through a hole in the external carotid artery. The nylon        thread is pushed into the ICA to the right MCA origin (20-23        mm). 90-120 minutes later the thread is pulled off, the animal        is closed and allowed to recover.    -   3. Permanent middle cerebral artery occlusion (MCAO)—occlusion        is permanent, unilateral-induced by electrocoagulation of MCA.        Both methods lead to focal brain ischemia of the ipsilateral        side of the brain cortex leaving the contralateral side intact        (control). The left MCA is exposed via a temporal craniectomy,        as described for rats by Tamura A. et al., J Cereb Blood Flow        Metab. 1981; 1:53-60. The MCA and its lenticulostriatal branch        are occluded proximally to the medial border of the olfactory        tract with microbipolar coagulation. The wound is sutured, and        animals returned to their home cage in a room warmed at 26° C.        to 28° C. The temperature of the animals is maintained all the        time with an automatic thermostat.

5. Acute Renal Failure (ARF)

Testing active siRNA for treating ARF may be done using sepsis-inducedARF or ischemia-reperfusion-induced ARF.

1. Sepsis Induced ARF

Two predictive animal models of sepsis-induced ARF are described byMiyaji T, Hu X, Yuen P S, Muramatsu Y, Iyer S, Hewitt S M, Star R A,2003, Ethyl pyruvate decreases sepsis-induced acute renal failure andmultiple organ damage in aged mice, Kidney Int. November; 64(5):1620-31.These two models are lipopolysaccharide administration and cecalligation puncture in mice, preferably in aged mice.

2. Ischemia-Reperfusion-Induced ARF

This predictive animal model is described by Kelly K J, Plotkin Z,Vulgamott S L, Dagher P C, 2003 January. P53 mediates the apoptoticresponse to GTP depletion after renal ischemia-reperfusion: protectiverole of a p53 inhibitor, J Am Soc Nephrol.; 14(1): 128-38.

Ischemia-reperfusion injury is induced in rats following 45 minutesbilateral kidney arterial clamp and subsequent release of the clamp toallow 24 hours of reperfusion. anti RTP801L siRNA or GFP siRNA (negativecontrol) are injected into the jugular vein 2 hrs prior to and 30minutes following the clamp. Additional siRNA is given via the tail veinat 4 and 8 hrs after the clamp. ARF progression is monitored bymeasurement of serum creatinine levels before and 24 hrs post surgery.At the end of the experiment, the rats are perfused via an indwellingfemoral line with warm PBS followed by 4% paraformaldehyde. The leftkidneys are removed and stored in 4% paraformaldehyde for subsequenthistological analysis. Acute renal failure is frequently defined as anacute increase of the serum creatinine level from baseline. An increaseof at least 0.5 mg per dL or 44.2 μmol per L of serum creatinine isconsidered as an indication for acute renal failure. Serum creatinine ismeasured at time zero before the surgery and at 24 hours post ARFsurgery. siRNA to 801L prevents production of ARF in this model

To study the distribution of siRNA in the rat kidney, Cy3-labeled 19-merblunt-ended siRNA molecules (2 mg/kg) having alternating O-methylmodification in the sugar residues were administered iv for 3-5 min,after which in vivo imaging was conducted using two-photon confocalmicroscopy. The confocal microscopy analysis revealed that the majorityof siRNA in the kidneys is concentrated in the endosomal compartment ofproximal tubular cells. Both endosomal and cytoplasmic siRNAfluorescence were relatively stable during the first 2 hrs post deliveryand disappeared at 24 hrs.

The expression of RTP801L during ischemia-reperfurion induced ARF wasexamined in rat kidneys. In both kidney regions, cortex and medulla,RTP801L transcript level is decreased in the ARF-10 hr group relative tothe control group transcript level. RTP801L transcript level is alsoelevated (up-regulated) in the kidney medulla, 3 and 6 hrs following theARF operation (bilateral renal artery clamp).

Example 5

Preparation of siRNAs

Using proprietary algorithms and the known sequence of gene RTP801L (SEQID NO:1), the sequences of many potential siRNAs were generated. siRNAmolecules according to the above specifications were preparedessentially as described herein.

The siRNAs of the present invention can be synthesized by any of themethods which are well-known in the art for synthesis of ribonucleic (ordeoxyribonucleic) oligonucleotides. For example, a commerciallyavailable machine (available, inter alia, from Applied Biosystems) canbe used; the oligonucleotides are prepared according to the sequencesdisclosed herein. Overlapping pairs of chemically synthesized fragmentscan be ligated using methods well known in the art (e.g., see U.S. Pat.No. 6,121,426). The strands are synthesized separately and then areannealed to each other in the tube. Then, the double-stranded siRNAs areseparated from the single-stranded oligonucleotides that were notannealed (e.g. because of the excess of one of them) by HPLC. Inrelation to the siRNAs or siRNA fragments of the present invention, twoor more such sequences can be synthesized and linked together for use inthe present invention.

The siRNA molecules of the invention may be synthesized by proceduresknown in the art e.g. the procedures as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684; andWincott et al., 1997, Methods Mol. Bio., 74, 59, and may make use ofcommon nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. Themodified (e.g. 2′-O-methylated) nucleotides and unmodified nucleotidesare incorporated as desired. Alternatively, the nucleic acid moleculesof the present invention can be synthesized separately and joinedtogether post-synthetically, for example, by ligation (Moore et al.,1992, Science 256, 9923; Draper et al., International PCT publicationNo. WO93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247;Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al.,1997, Bioconjugate Chem. 8, 204), or by hybridization followingsynthesis and/or deprotection. The siRNA molecules of the invention canalso be synthesized via a tandem synthesis methodology, as described inUS patent application publication No. US2004/0019001 (McSwiggen) whereinboth siRNA strands are synthesized as a single contiguousoligonucleotide fragment or strand separated by a cleavable linker whichis subsequently cleaved to provide separate siRNA fragments or strandsthat hybridize and permit purification of the siRNA duplex. The linkercan be a polynucleotide linker or a non-nucleotide linker.

For further information, see PCT publication No. WO 2004/015107(ATUGEN).

As described above, the siRNAs consisting of the sequences set forth inTable A (below) were constructed such that alternate sugars have2′-O-methyl modification i.e. alternate nucleotides were thus modified.In these preferred embodiments, in one strand of the siRNA the modifiednucleotides were numbers 1,3,5,7,9,11,13,15,17 and 19 and in theopposite strand the modified nucleotides were numbers2,4,6,8,10,12,14,16 and 18. Thus these siRNAs are blunt-ended 19-mer RNAmolecules with alternate 2-0′-methyl modifications as described above.

TABLE A human 34222182 mouse chimpanzee dog No Sp-Source Sense siRNAAntiSense siRNA cds = 204-785 31541838 rat 62644440 55622975 74002279 1human GCCAGUGUUCUAACAAACU AGUUUGUUAGAACACUGGC [2242-2260](19/19) — — — —2 human UAGCCAGUGUUCUAACAAA UUUGUUAGAACACUGGCUA [2240-2258](19/19) — — —— 3 human GUGACUUCCUCACUCUAAU AUUAGAGUGAGGAAGUCAC [1385-1403](19/19) — —— — 4 human AGCCAGUGUUCUAACAAAC GUUUGUUAGAACACUGGCU [2241-2259](19/19) —— — — 5 human GAGUGAAUGAUGAAUACCU AGGUAUUCAUCAUUCACUC [1577-1595](19/19)— — — — 6 human GACUUCCUCACUCUAAUGU ACAUUAGAGUGAGGAAGUC[1387-1405](19/19) — — — — 7 human GUUCUAACAAACUAAACUCGAGUUUAGUUUGUUAGAAC [2248-2266](19/19) — — [531-541](11/11) — 8 humanGAAUGAUGAAUACCUGUGA UCACAGGUAUUCAUCAUUC [1581-1599](19/19) — — — — 9human UCCUCACUCUAAUGUUUUA UAAAACAUUAGAGUGAGGA [1391-1409](19/19) — — — —10 human, mouse, GCCAGAAUUUGGUUAAAAU AUUUUAACCAAAUUCUGGC[364-382](19/19) [362-380](19/19) [243-261](19/19) [484-502](19/19) —rat, chimpanzee 11 human ACGGGUCAAUUUACGAAGU ACUUCGUAAAUUGACCCGU[1809-1827](19/19) — — — — 12 human UCCAUUGAGUGAAUGAUGAUCAUCAUUCACUCAAUGGA [1571-1589](19/19) — — — — 13 humanUUCCUCACUCUAAUGUUUU AAAACAUUAGAGUGAGGAA [1390-1408](19/19) — — — — 14human GCACCCAGAUUUUUUCCAC GUGGAAAAAAUCUGGGUGC [1901-1919](19/19) — — — —15 human GUGGUGCCAUUUCAGUAAC GUUACUGAAAUGGCACCAC [1428-1446](19/19) — —— — 16 human CCUCACUCUAAUGUUUUAA UUAAAACAUUAGAGUGAGG [1392-1410](19/19)— — — — 17 human CUUCCUCACUCUAAUGUUU AAACAUUAGAGUGAGGAAG[1389-1407](19/19) — — — — 18 human GGCUUUUUUUUCUCUAAGUACUUAGAGAAAAAAAAGCC [1153-1171](19/19) — — — — 19 humanUCCCAUUUUUGUACAGAAU AUUCUGUACAAAAAUGGGA [1005-1023](19/19) — — — — 20human GAGAAGUGAUUCAAAAUAG CUAUUUUGAAUCACUUCUC [967-985](19/19) — — — —21 human GGAGAAGUGAUUCAAAAUA UAUUUUGAAUCACUUCUCC [966-984](19/19) — — —— 22 human GUCAGCUAAAGUCAUUUGU ACAAAUGACUUUAGCUGAC [841-859](19/19) — —— — 23 human, chimpanzee CCGGCCAGCAUUUCAGAAU AUUCUGAAAUGCUGGCCGG[237-255](19/19) [235-251](17/17) [117-132](16/16) [357-375](19/19) — 24human, chimpanzee, AUGCUGGAGAACUGUCUGU ACAGACAGUUCUCCAGCAU[381-399](19/19) [383-397](15/15) [264-278](15/15) [501-519](19/19)[363-381] dog (19/19) 25 human, chimpanzee, AAAUGCUGGAGAACUGUCUAGACAGUUCUCCAGCAUUU [379-397](19/19) [377-395](18/19) [258-276](18/19)[499-517](19/19) [361-379] dog (19/19) 26 human, chimpanzeeUGGUUAAAAUGCUGGAGAA UUCUCCAGCAUUUUAACCA [373-391](19/19)[371-389](18/19) [252-270](18/19) [493-511](19/19) [359-373] (15/15) 27human, chimpanzee UUGGUUAAAAUGCUGGAGA UCUCCAGCAUUUUAACCAA[372-390](19/19) [370-388](18/19) [251-269](18/19) [492-510](19/19)[354-372] (18/19) 28 human UAGCUCCACUUCACAUGCU AGCAUGUGAAGUGGAGCUA[2118-2136](19/19) — — — — 29 human AGCCUCCACUCAACAAUGUACAUUGUUGAGUGGAGGCU [2023-2041](19/19) — — — — 30 humanACCCAGAUUUUUUCCACCU AGGUGGAAAAAAUCUGGGU [1903-1921](19/19) — — — — 31human UGCACCCAGAUUUUUUCCA UGGAAAAAAUCUGGGUGCA [1900-1918](19/19) — — — —32 human AAACGGGUCAAUUUACGAA UUCGUAAAUUGACCCGUUU [1807-1825](19/19) — —— — 33 human AUGAUGAAUACCUGUGAGG CCUCACAGGUAUUCAUCAU [1583-1601](19/19)— — — — 34 human UUGAGUGAAUGAUGAAUAC GUAUUCAUCAUUCACUCAA[1575-1593](19/19) — — — — 35 human CGGCAAUAAUGGAACUGCUAGCAGUUCCAUUAUUGCCG [1353-1371](19/19) — — — — 36 humanGCCUAUCAAAACUUCCAAA UUUGGAAGUUUUGAUAGGC [1218-1236](19/19) — — —[1182-1194] (13/13) 37 human UGGCUUUUUUUUCUCUAAG CUUAGAGAAAAAAAAGCCA[1152-1170](19/19) — — — — 38 human GCCCAUUUGAGUUUUACAUAUGUAAAACUCAAAUGGGC [1057-1075](19/19) — — — — 39 human, chimpanzeeGGCCAGCAUUUCAGAAUUG CAAUUCUGAAAUGCUGGCC [239-257](19/19)[237-251](15/15) [118-132](15/15) [359-377](19/19) — 40human, chimpanzee CGGCCAGCAUUUCAGAAUU AAUUCUGAAAUGCUGGCCG[238-256](19/19) [236-251](16/16) [117-132](16/16) [358-376](19/19) — 41human GCGUCGUACCUACUUUUGA UCAAAAGUAGGUACGACGC [589-607](19/19) — —[711-727](16/17) — 42 human UAGCGUCGUACCUACUUUU AAAAGUAGGUACGACGCUA[587-605](19/19) — — — — 43 human, rat, dog AUGCACGUGAACUUGGAAAUUUCCAAGUUCACGUGCAU [525-543](19/19) [523-539](17/17) [404-422](19/19)[645-663](18/19) [507-525] (19/19) 44 human UUGUCCUUUUUCCACUAACGUUAGUGGAAAAAGGACAA [2441-2459](19/19) — — — — 45 humanGUUGUCCUUUUUCCACUAA UUAGUGGAAAAAGGACAAC [2440-2458](19/19) — — — — 46human CUGUUGUCCUUUUUCCACU AGUGGAAAAAGGACAACAG [2438-2456](19/19) — — — —47 human, chimpanzee, CUGGAGAACUGUCUGUCCA UGGACAGACAGUUCUCCAG[384-402](19/19) [383-400](18/18) [264-281](18/18) [504-522](19/19)[366-384] dog (19/19) 48 human GCCAAUCUUUAUAGAAUUG CAAUUCUAUAAAGAUUGGC[2294-2312](19/19) — — — — 49 human GUUCAAAUUAGCCAGUGUUAACACUGGCUAAUUUGAAC [2232-2250](19/19) — — — — 50 human, mouse,UGCCAGAAUUUGGUUAAAA UUUUAACCAAAUUCUGGCA [363-381](19/19)[361-379](19/19) [242-260](19/19) [483-501](19/19) — rat, chimpanzee 51human CCCAGAUUUUUUCCACCUU AAGGUGGAAAAAAUCUGGG [1904-1922](19/19) — — — —52 human CACCCAGAUUUUUUCCACC GGUGGAAAAAAUCUGGGUG [1902-1920](19/19) — —— — 53 human CAAUUUACGAAGUCUGCAU AUGCAGACUUCGUAAAUUG [1815-1833](19/19)— — — — 54 human GGGUCAAUUUACGAAGUCU AGACUUCGUAAAUUGACCC[1811-1829](19/19) — — — — 55 human AACGGGUCAAUUUACGAAGCUUCGUAAAUUGACCCGUU [1808-1826](19/19) — — — — 56 humanUGAAACGGGUCAAUUUACG CGUAAAUUGACCCGUUUCA [1805-1823](19/19) — — — — 57human UGGUGCCAUUUCAGUAACC GGUUACUGAAAUGGCACCA [1429-1447](19/19) — — — —58 human UGGAACUGCUUCACUGUUU AAACAGUGAAGCAGUUCCA [1362-1380](19/19) — —— [1333-1343] (11/11) 59 human AAGGUAGGAUUAAGUAGGU ACCUACUUAAUCCUACCUU[1286-1304](19/19) — — — — 60 human CAGGAAGGUAGGAUUAAGUACUUAAUCCUACCUUCCUG [1282-1300](19/19) — — — — 61 humanAGCCUAUCAAAACUUCCAA UUGGAAGUUUUGAUAGGCU [1217-1235](19/19) — — —[1182-1194] (13/13) 62 human AACCAGAUUUGCCUAUUUU AAAAUAGGCAAAUCUGGUU[1123-1141](19/19) — — — — 63 human GUACAGAAUUGAAUGGGAUAUCCCAUUCAAUUCUGUAC [1015-1033](19/19) — — — — 64 humanAUCCCAUUUUUGUACAGAA UUCUGUACAAAAAUGGGAU [1004-1022](19/19) — — — — 65human CAGCUAAAGUCAUUUGUAG CUACAAAUGACUUUAGCUG [843-861](19/19)[845-856](12/12) — — [834-844] (11/11) 66 human AUGAUUGGGUAGUAAAACUAGUUUUACUACCCAAUCAU [813-831](19/19) — — — — 67 humanAGGGUCCUAAAAAGGGAAA UUUCCCUUUUUAGGACCCU [776-794](19/19) — — — — 68human, rat, dog ACGUGAACUUGGAAAUUGA UCAAUUUCCAAGUUCACGU [529-547](19/19)[527-545](18/19) [408-426](19/19) [651-667](17/17) [511-529] (19/19) 69human, mouse, GAAUUGCUCAAGAUGUCCU AGGACAUCUUGAGCAAUUC [463-481](19/19)[461-479](19/19) [342-360](18/19) [583-601](19/19) [447-463] chimpanzee(17/17) 70 human, mouse, GAGAAUUGCUCAAGAUGUC GACAUCUUGAGCAAUUCUC[461-479](19/19) [459-477](19/19) — [581-599](19/19) [447-461]chimpanzee (15/15) 71 human, mouse, AGAGAAUUGCUCAAGAUGUACAUCUUGAGCAAUUCUCU [460-478](19/19) [458-476](19/19) — [580-598](19/19)[442-460] chimpanzee (18/19) 72 human, mouse,  CCAGAGAAUUGCUCAAGAUAUCUUGAGCAAUUCUCUGG [458-476](19/19) [456-474](19/19) [337-355](18/19)[578-596](19/19) [440-458] chimpanzee (18/19) 73 human, mouse,CCCAGAGAAUUGCUCAAGA UCUUGAGCAAUUCUCUGGG [457-475](19/19)[455-473](19/19) [336-354](18/19) [577-595](19/19) [439-457] chimpanzee(18/19) 74 human, chimpanzee, AACUGUCUGUCCAAAUCAA UUGAUUUGGACAGACAGUU[390-408](19/19) [388-406](18/19) [269-287](18/19) [510-528](19/19)[372-390] dog (19/19) 75 human UGUCCUUUUUCCACUAACA UGUUAGUGGAAAAAGGACA[2442-2460](19/19) — — — — 76 human GAACUGUUGUCCUUUUUCCGGAAAAAGGACAACAGUUC [2435-2453](19/19) — — — — 77 human, chimpanzee,GGAGAACUGUCUGUCCAAA UUUGGACAGACAGUUCUCC [386-404](19/19)[384-400](17/17) [265-282](18/18) [506-524](19/19) [368-386] dog (19/19)78 human CAGUGUUCUAACAAACUAA UUAGUUUGUUAGAACACUG [2244-2262](19/19) — —— — 79 human AAAUUAGCCAGUGUUCUAA UUAGAACACUGGCUAAUUU [2236-2254](19/19)— — — — 80 human GACCUAAAAUGUCACUGUU AACAGUGACAUUUUAGGUC[2216-2234](19/19) — — — — 81 human, chimpanzee UUGCCAGAAUUUGGUUAAAUUUAACCAAAUUCUGGCAA [362-380](19/19) [361-378](18/18) [242-259](18/18)[482-500](19/19) — 82 human UGGAUAAGGAGCUUAUUCA UGAAUAAGCUCCUUAUCCA[2080-2098](19/19) — — — — 83 human AGCAAGGCUUUCAUAUCCUAGGAUAUGAAAGCCUUGCU [2049-2067](19/19) — — — — 84 humanCUCCACUCAACAAUGUUCA UGAACAUUGUUGAGUGGAG [2026-2044](19/19) — — — — 85human UUAGCCUCCACUCAACAAU AUUGUUGAGUGGAGGCUAA [2021-2039](19/19) — — — —86 human AGAGAAUUUAGCCUCCACU AGUGGAGGCUAAAUUCUCU [2014-2032](19/19) — —— — 87 human AGAUCAUUAUCUCUUUCCU AGGAAAGAGAUAAUGAUCU [1981-1999](19/19)— — — — 88 human GGCCUUAUUUUUUGUCUUA UAAGACAAAAAAUAAGGCC[1950-1968](19/19) — — — — 89 human CAGAUUUUUUCCACCUUGGCCAAGGUGGAAAAAAUCUG [1906-1924](19/19) — — — — 90 humanCCAGAUUUUUUCCACCUUG CAAGGUGGAAAAAAUCUGG [1905-1923](19/19) — — — — 91human GCCUAGAGAAUGAAACUCA UGAGUUUCAUUCUCUAGGC [1862-1880](19/19) — — — —92 human UACGAAGUCUGCAUUGGCU AGCCAAUGCAGACUUCGUA [1820-1838](19/19) — —— — 93 human CGGGUCAAUUUACGAAGUC GACUUCGUAAAUUGACCCG [1810-1828](19/19)— — — — 94 human GAAACGGGUCAAUUUACGA UCGUAAAUUGACCCGUUUC[1806-1824](19/19) — — — — 95 human GUCCCUCUCUGAUUCACUUAAGUGAAUCAGAGAGGGAC [1626-1644](19/19) [1278-1288] — — — (11/11) 96human GAGAGGGGACUCCUAAGAA UUCUUAGGAGUCCCCUCUC [78-96](19/19) — — — — 97human GAAGGUAGGAUUAAGUAGG CCUACUUAAUCCUACCUUC [1285-1303](19/19) — — — —98 human UAGCCUAUCAAAACUUCCA UGGAAGUUUUGAUAGGCUA [1216-1234](19/19) — —— [1182-1194] (13/13) 99 human CCAUUUUUGUACAGAAUUG CAAUUCUGUACAAAAAUGG[1007-1025](19/19) — — — — 100 human UAGAUCCCAUUUUUGUACAUGUACAAAAAUGGGAUCUA [1001-1019](19/19) — — — — 101 humanGCAGCUAACAGGCUGAUUU AAAUCAGCCUGUUAGCUGC [937-955](19/19) — — — — 102human GUGUUUCACAUUCAUAGCA UGCUAUGAAUGUGAAACAC [915-933](19/19) — — — —103 human GUCCUAAAAAGGGAAAAUA UAUUUUCCCUUUUUAGGAC [779-797](19/19) — — —— 104 human GGGUCCUAAAAAGGGAAAA UUUUCCCUUUUUAGGACCC [777-795](19/19) — —— — 105 human AAGGGUCCUAAAAAGGGAA UUCCCUUUUUAGGACCCUU [775-793](19/19) —— — — 106 human CAGGGACUUUUUCUUUAGU ACUAAAGAAAAAGUCCCUG [653-671](19/19)— — [773-789](17/17) — 107 human, rat, dog UGCACGUGAACUUGGAAAUAUUUCCAAGUUCACGUGCA [526-544](19/19) [524-539](16/16) [405-423](19/19)[646-664](18/19) [508-526] (19/19) 108 human, mouse, GUGUUAUGCACGUGAACUUAAGUUCACGUGCAUAACAC [520-538](19/19) [518-536](19/19) [399-417](19/19) —[502-520] rat, dog (19/19) 109 human, mouse GUUGUGUUAUGCACGUGAAUUCACGUGCAUAACACAAC [517-535](19/19) [515-533](19/19) [398-414](17/17)[637-655](18/19) [501-517] (17/17) 110 human GAGGUUGUGUUAUGCACGUACGUGCAUAACACAACCUC [514-532](19/19) [514-530](17/17) [398-411](14/14)[634-649](16/16) [496-514] (18/19) 111 human, mouse, GACCCAGAGAAUUGCUCAAUUGAGCAAUUCUCUGGGUC [455-473](19/19) [453-471](19/19) [334-348](15/15)[575-593](19/19) — chimpanzee 112 human, chimpanzee AAGCAAACUAAACUUGGUUAACCAAGUUUAGUUUGCUU [408-426](19/19) — — [528-546](19/19) — 113human, chimpanzee CCAAAUCAAAGCAAACUAA UUAGUUUGCUUUGAUUUGG[400-418](19/19) [402-413](12/12) [279-294](15/16) [520-538](19/19)[382-397] (16/16) 114 human GGAAGGCUGUUAAAUUAAU AUUAAUUUAACAGCCUUCC[2512-2530](19/19) — — — — 115 human UGCCUGUUAUGCUUACAAAUUUGUAAGCAUAACAGGCA [2478-2496](19/19) — — — — 116 humanUUGCCUGUUAUGCUUACAA UUGUAAGCAUAACAGGCAA [2477-2495](19/19) — — — — 117human UGACUCUCUUGCCUGUUAU AUAACAGGCAAGAGAGUCA [2469-2487](19/19) — — — —118 human GUCCUUUUUCCACUAACAG CUGUUAGUGGAAAAAGGAC [2443-2461](19/19) — —— — 119 human UAGAACUGUUGUCCUUUUU AAAAAGGACAACAGUUCUA [2433-2451](19/19)— — — — 120 human GUAGAACUGUUGUCCUUUU AAAAGGACAACAGUUCUAC[2432-2450](19/19) — — — — 121 human, chimpanzee, GAGAACUGUCUGUCCAAAUAUUUGGACAGACAGUUCUC [387-405](19/19) [385-400](16/16) [266-282](17/17)[507-525](19/19) [369-387] dog (19/19) 122 human GCCAAGAUAAAUCAAUGUUAACAUUGAUUUAUCUUGGC [2314-2332](19/19) — — — — 123 humanACAAAGCCAAUCUUUAUAG CUAUAAAGAUUGGCUUUGU [2289-2307](19/19) — — — — 124human AUGUCACUGUUCAAAUUAG CUAAUUUGAACAGUGACAU [2224-2242](19/19) — — — —125 human GUGAUCCUGUUACUGAUAC GUAUCAGUAACAGGAUCAC [2190-2208](19/19) — —— — 126 human, chimpanzee GAAUUUGGUUAAAAUGCUG CAGCAUUUUAACCAAAUUC[368-386](19/19) [366-381](16/16) [247-262](16/16) [488-506](19/19) —127 human AGGCUUUCAUAUCCUUGCU AGCAAGGAUAUGAAAGCCU [2053-2071](19/19) — —— — 128 human CAGCAAGGCUUUCAUAUCC GGAUAUGAAAGCCUUGCUG [2048-2066](19/19)— — — — 129 human GCCUCCACUCAACAAUGUU AACAUUGUUGAGUGGAGGC[2024-2042](19/19) — — — — 130 human GAAUUUAGCCUCCACUCAAUUGAGUGGAGGCUAAAUUC [2017-2035](19/19) — — — — 131 humanGAGAGAAUUUAGCCUCCAC GUGGAGGCUAAAUUCUCUC [2013-2031](19/19) — — — — 132human UAGAUCAUUAUCUCUUUCC GGAAAGAGAUAAUGAUCUA [1980-1998](19/19) — — — —133 human AGGCCUUAUUUUUUGUCUU AAGACAAAAAAUAAGGCCU [1949-1967](19/19) — —— — 134 human AAGGCCUUAUUUUUUGUCU AGACAAAAAAUAAGGCCUU [1948-1966](19/19)— — — — 135 human GCAUGCACCCAGAUUUUUU AAAAAAUCUGGGUGCAUGC[1897-1915](19/19) — — — — 136 human GGUCAAUUUACGAAGUCUGCAGACUUCGUAAAUUGACC [1812-1830](19/19) — — — — 137 humanGGGCUUUUCUGGGAAUUGA UCAAUUCCCAGAAAAGCCC [1725-1743](19/19) — — — — 138human AUACCUGUGAGGAUAGGAA UUCCUAUCCUCACAGGUAU [1590-1608](19/19) — — — —139 human ACUCUUCCAUUGAGUGAAU AUUCACUCAAUGGAAGAGU [1566-1584](19/19) — —— — 140 human, chimpanzee GGGAUUAUGUUGUUCCUGA UCAGGAACAACAUAAUCCC[307-325](19/19) [305-323](18/19) — [427-445](19/19) — 141 humanUGCCAUUUCAGUAACCACG CGUGGUUACUGAAAUGGCA [1432-1450](19/19) — — — — 142human UGUGGUGCCAUUUCAGUAA UUACUGAAAUGGCACCACA [1427-1445](19/19) — — — —143 human AGCUUGUGGUGCCAUUUCA UGAAAUGGCACCACAAGCU [1423-1441](19/19) — —— — 144 human CUCUAAUGUUUUAAAGAGG CCUCUUUAAAACAUUAGAG [1397-1415](19/19)— — — — 145 human GAACUGCUUCACUGUUUCU AGAAACAGUGAAGCAGUUC[1364-1382](19/19) — — — [1333-1345] (13/13) 146 humanGGAACUGCUUCACUGUUUC GAAACAGUGAAGCAGUUCC [1363-1381](19/19) — — —[1333-1344] (12/12) 147 human ACGGCAAUAAUGGAACUGC GCAGUUCCAUUAUUGCCGU[1352-1370](19/19) — — — — 148 human ACCCUAGGUAAGAGUAAAUAUUUACUCUUACCUAGGGU [1323-1341](19/19) — — — — 149 humanCUCUAAGUUUUCAGAGGAU AUCCUCUGAAAACUUAGAG [1164-1182](19/19) — — — — 150human GCUUGGUAAUAGACUAUAU AUAUAGUCUAUUACCAAGC [1103-1121](19/19) — — — —151 human AGGCUUGGUAAUAGACUAU AUAGUCUAUUACCAAGCCU [1101-1119](19/19) — —— — 152 human GAGUUUUACAUUUGAUUCC GGAAUCAAAUGUAAAACUC [1065-1083](19/19)— — — — 153 human GAAGCCCAUUUGAGUUUUA UAAAACUCAAAUGGGCUUC[1054-1072](19/19) — — — — 154 human, chimpanzee GAGCCUGCUAAGUGAUUUUAAAAUCACUUAGCAGGCUC [281-299](19/19) [283-296](14/14) [164-177](14/14)[401-419](19/19) [263-281] (18/19) 155 human UGUACAGAAUUGAAUGGGAUCCCAUUCAAUUCUGUACA [1014-1032](19/19) — — — — 156 humanUUGUACAGAAUUGAAUGGG CCCAUUCAAUUCUGUACAA [1013-1031](19/19) — — — — 157human GUGAUUCAAAAUAGUGUAG CUACACUAUUUUGAAUCAC [972-990](19/19) — — — —158 human UUGGAGAAGUGAUUCAAAA UUUUGAAUCACUUCUCCAA [964-982](19/19) — — —— 159 human CAGGCUGAUUUUCUGGCCU AGGCCAGAAAAUCAGCCUG [945-963](19/19)[939-949](11/11) — — — 160 human GCUAACAGGCUGAUUUUCU AGAAAAUCAGCCUGUUAGC[940-958](19/19) [939-949](11/11) — — — 161 human GCUAAAGUCAUUUGUAGUUAACUACAAAUGACUUUAGC [845-863](19/19) [845-858](14/14) — — [834-845](12/12) 162 human UAGUCAGCUAAAGUCAUUU AAAUGACUUUAGCUGACUA[839-857](19/19) — — — — 163 human CUAGUCAGCUAAAGUCAUUAAUGACUUUAGCUGACUAG [838-856](19/19) — — — — 164 humanUGAUUGGGUAGUAAAACUA UAGUUUUACUACCCAAUCA [814-832](19/19) — — — — 165human, chimpanzee GCAUUUCAGAAUUGCUGGA UCCAGCAAUUCUGAAAUGC[244-262](19/19) — — [364-382](19/19) — 166 human GAAGGGUCCUAAAAAGGGAUCCCUUUUUAGGACCCUUC [774-792](19/19) — — — — 167 humanGGUUUCAGGAGAACUCUGA UCAGAGUUCUCCUGAAACC [690-708](19/19) — — — — 168human UCCUCUGGUUUCAGGAGAA UUCUCCUGAAACCAGAGGA [684-702](19/19) — — — —169 human AGGGACUUUUUCUUUAGUA UACUAAAGAAAAAGUCCCU [654-672](19/19) — —[774-789](16/16) — 170 human, chimpanzee CUACUUUUGAGCUUACACUAGUGUAAGCUCAAAAGUAG [598-616](19/19) — — [718-736](19/19) — 171 humanCUAGCGUCGUACCUACUUU AAAGUAGGUACGACGCUAG [586-604](19/19) — — — — 172human, mouse, GUAAAAAGCUGGAUAGGAU AUCCUAUCCAGCUUUUUAC [556-574](19/19)[554-572](19/19) [435-453](19/19) [676-694](19/19) [538-552]rat, chimpanzee (15/15) 173 human, mouse, UUAUGCACGUGAACUUGGAUCCAAGUUCACGUGCAUAA [523-541](19/19) [521-539](19/19) [402-420](19/19)[643-661](18/19) [505-523] rat, dog (19/19) 174 human, mouseGGUUGUGUUAUGCACGUGA UCACGUGCAUAACACAACC [516-534](19/19)[514-532](19/19) [398-413](16/16) [636-654](18/19) [501-516] (16/16) 175human GCGAGGUUGUGUUAUGCAC GUGCAUAACACAACCUCGC [512-530](19/19)[514-528](15/15) [398-409](12/12) [632-649](18/18) [494-512] (18/19) 176human, chimpanzee AAAGCAAACUAAACUUGGU ACCAAGUUUAGUUUGCUUU[407-425](19/19) — — [527-545](19/19) — 177 human CUGUUAUGCUUACAAAAUGCAUUUUGUAAGCAUAACAG [2481-2499](19/19) — — — — 178 humanUCCUUUUUCCACUAACAGU ACUGUUAGUGGAAAAAGGA [2444-2462](19/19) — — — — 179human CAAUCUUUAUAGAAUUGGG CCCAAUUCUAUAAAGAUUG [2296-2314](19/19) — — — —180 human CCAAUCUUUAUAGAAUUGG CCAAUUCUAUAAAGAUUGG [2295-2313](19/19) — —— — 181 human AUACUACAAAGCCAAUCUU AAGAUUGGCUUUGUAGUAU [2284-2302](19/19)[1571-1581] — — — (11/11) 182 human CCAGUGUUCUAACAAACUAUAGUUUGUUAGAACACUGG [2243-2261](19/19) — — — — 183 humanACUGUUCAAAUUAGCCAGU ACUGGCUAAUUUGAACAGU [2229-2247](19/19) — — — — 184human CCUAAAAUGUCACUGUUCA UGAACAGUGACAUUUUAGG [2218-2236](19/19) — — — —185 human UGACCUAAAAUGUCACUGU ACAGUGACAUUUUAGGUCA [2215-2233](19/19) — —— — 186 human UAAGUGACCUAAAAUGUCA UGACAUUUUAGGUCACUUA [2211-2229](19/19)— — — — 187 human CUAUAAGUGACCUAAAAUG CAUUUUAGGUCACUUAUAG[2208-2226](19/19) — — — — 188 human GUGUGAUCCUGUUACUGAUAUCAGUAACAGGAUCACAC [2188-2206](19/19) — — — — 189 humanCCACUUCACAUGCUGGAGA UCUCCAGCAUGUGAAGUGG [2123-2141](19/19) — — — — 190human GGCUUUCAUAUCCUUGCUG CAGCAAGGAUAUGAAAGCC [2054-2072](19/19) — — — —191 human GCAAGGCUUUCAUAUCCUU AAGGAUAUGAAAGCCUUGC [2050-2068](19/19) — —— — 192 human CACUCAACAAUGUUCAAUU AAUUGAACAUUGUUGAGUG [2029-2047](19/19)— — — — 193 human UAGCCUCCACUCAACAAUG CAUUGUUGAGUGGAGGCUA[2022-2040](19/19) — — — — 194 human GUAGAUCAUUAUCUCUUUCGAAAGAGAUAAUGAUCUAC [1979-1997](19/19) — — — — 195 humanCCACCUUGGAUACCUGUCA UGACAGGUAUCCAAGGUGG [1916-1934](19/19) — — — — 196human AUGCAUGCACCCAGAUUUU AAAAUCUGGGUGCAUGCAU [1895-1913](19/19) — — — —197 human UUGAAACGGGUCAAUUUAC GUAAAUUGACCCGUUUCAA [1804-1822](19/19) — —— — 198 human, chimpanzee UCAACGAGGUAAUAUUUGA UCAAAUAUUACCUCGUUGA[334-352](19/19) — — [454-472](19/19) — 199 human, chimpanzeeACCUCAACGAGGUAAUAUU AAUAUUACCUCGUUGAGGU [331-349](19/19)[329-341](13/13) [210-222](13/13) [451-469](19/19) — 200human, chimpanzee CCAACCUCAACGAGGUAAU AUUACCUCGUUGAGGUUGG[328-346](19/19) [326-341](16/16) [207-222](16/16) [448-466](19/19) —201 human GUGCUUAAUCUCAGAUGAA UUCAUCUGAGAUUAAGCAC [1674-1692](19/19) — —— — 202 human CUAGUCCCUCUCUGAUUCA UGAAUCAGAGAGGGACUAG [1623-1641](19/19)[1278-1288] — — — (11/11) 203 human AUGAAUACCUGUGAGGAUAUAUCCUCACAGGUAUUCAU [1586-1604](19/19) — — — — 204 humanAGAGGGGACUCCUAAGAAG CUUCUUAGGAGUCCCCUCU [79-97](19/19) — — — — 205 humanGAUUACUCUUCCAUUGAGU ACUCAAUGGAAGAGUAAUC [1562-1580](19/19) — — — — 206human UGAUUACUCUUCCAUUGAG CUCAAUGGAAGAGUAAUCA [1561-1579](19/19) — — — —207 human UAGUUGAUUACUCUUCCAU AUGGAAGAGUAAUCAACUA [1557-1575](19/19) — —— — 208 human GUAGUUGAUUACUCUUCCA UGGAAGAGUAAUCAACUAC [1556-1574](19/19)— — — — 209 human GUGUUGAAUACUGUCUUUA UAAAGACAGUAUUCAACAC[1497-1515](19/19) — — — — 210 human AAGCUCAGUUUCCCCUGUUAACAGGGGAAACUGAGCUU [1473-1491](19/19) — — — — 211 humanACCACGGUGUUGUUUUAGA UCUAAAACAACACCGUGGU [1445-1463](19/19) — — — — 212human GUGCCAUUUCAGUAACCAC GUGGUUACUGAAAUGGCAC [1431-1449](19/19) — — — —213 human GGUGCCAUUUCAGUAACCA UGGUUACUGAAAUGGCACC [1430-1448](19/19) — —— — 214 human CUGCUUCACUGUUUCUUGG CCAAGAAACAGUGAAGCAG [1367-1385](19/19)— — — [1333-1346] (14/14) 215 human AACUGCUUCACUGUUUCUUAAGAAACAGUGAAGCAGUU [1365-1383](19/19) — — — [1333-1346] (14/14) 216human CAAUAAUGGAACUGCUUCA UGAAGCAGUUCCAUUAUUG [1356-1374](19/19) — — — —217 human AGGUAAGAGUAAAUGAGAA UUCUCAUUUACUCUUACCU [1328-1346](19/19) — —— — 218 human GGAUUAAGUAGGUGAGUUU AAACUCACCUACUUAAUCC [1292-1310](19/19)— — — — 219 human GACUCAAAUUUGAAGGGUU AACCCUUCAAAUUUGAGUC[1257-1275](19/19) — — — — 220 human CAGAUUUGCCUAUUUUGAUAUCAAAAUAGGCAAAUCUG [1126-1144](19/19) — — — — 221 humanCCAGAUUUGCCUAUUUUGA UCAAAAUAGGCAAAUCUGG [1125-1143](19/19) — — — — 222human AUAUAAACCAGAUUUGCCU AGGCAAAUCUGGUUUAUAU [1118-1136](19/19) — — — —223 human GGCUUGGUAAUAGACUAUA UAUAGUCUAUUACCAAGCC [1102-1120](19/19) — —— 224 human UUCCACAAUUUGGUUUCAG CUGAAACCAAAUUGUGGAA [1080-1098](19/19) —— — — 225 human UUUGAUUCCACAAUUUGGU ACCAAAUUGUGGAAUCAAA[1075-1093](19/19) — — — — 226 human GGAAUAGGUAAGCAAAAGUACUUUUGCUUACCUAUUCC [1034-1052](19/19) — — — — 227 humanCAGAAUUGAAUGGGAUGGA UCCAUCCCAUUCAAUUCUG [1018-1036](19/19) — — — — 228human GAUCCCAUUUUUGUACAGA UCUGUACAAAAAUGGGAUC [1003-1021](19/19) — — — —229 human AGAUCCCAUUUUUGUACAG CUGUACAAAAAUGGGAUCU [1002-1020](19/19) — —— — 230 human GUGUAGAUUUUCUGCAUAG CUAUGCAGAAAAUCUACAC [985-1003](19/19)— — — — 231 human AGGCUGAUUUUCUGGCCUU AAGGCCAGAAAAUCAGCCU[946-964](19/19) [939-949](11/11) — — — 232 human CACAUUCAUAGCAACUGCAUGCAGUUGCUAUGAAUGUG [921-939](19/19) — — — — 233 humanCCCCACCUGCCCUAAAUAA UUAUUUAGGGCAGGUGGGG [866-884](19/19) — — — — 234human AGCUAAAGUCAUUUGUAGU ACUACAAAUGACUUUAGCU [844-862](19/19)[845-857](13/13) — — [834-845] (12/12) 235 human UCAGCUAAAGUCAUUUGUAUACAAAUGACUUUAGCUGA [842-660](19/19) [845-855](11/11) — — — 236 humanCAGCUAGUCAGCUAAAGUC GACUUUAGCUGACUAGCUG [835-853](19/19) — — — — 237human UGGGUAGUAAAACUAUUCA UGAAUAGUUUUACUACCCA [818-836](19/19) — — — —238 human GAUUAUUUCAUGAUUGGGU ACCCAAUCAUGAAAUAAUC [804-822](19/19) — — —— 239 human GGUCCUAAAAAGGGAAAAU AUUUUCCCUUUUUAGGACC [778-796](19/19) — —— — 240  human, chimpanzee CAGCAUUUCAGAAUUGCUG CAGCAAUUCUGAAAUGCUG[242-260](19/19) [240-251](12/12) [121-139](18/19) [362-360](19/19) —241  human, chimpanzee GCCAGCAUUUCAGAAUUGC GCAAUUCUGAAAUGCUGGC[240-258](19/19) [238-251](14/14) [119-137](18/19) [360-378](19/19) —242 human AGAACUCUGAUCCUCAGCU AGCUGAGGAUCAGAGUUCU [699-717](19/19) —[579-596](18/18) — — 243 human UAAGAAGCCACCUGCCUGU ACAGGCAGGUGGCUUCUUA[91-109](19/19) — — — — 244 human CUCCUCUGGUUUCAGGAGAUCUCCUGAAACCAGAGGAG [683-701](19/19) — — — — 245 humanGGGACUUUUUCUUUAGUAG CUACUAAAGAAAAAGUCCC [655-673](19/19) — —[775-789](15/15) — 246 human, chimpanzee AGCUUACACUUGUGUUUAAUUAAACACAAGUGUAAGCU [607-625](19/19) [613-623](11/11) — [727-745](19/19)[589-607] (18/19) 247 human GUCGUACCUACUUUUGAGC GCUCAAAAGUAGGUACGAC[591-609](19/19) — — [711-729](18/19) — 248 human AGCGUCGUACCUACUUUUGCAAAAGUAGGUACGACGCU [588-606](19/19) — — — — 249 human, rat,AAGCUGGAUAGGAUUGUGU ACACAAUCCUAUCCAGCUU [561-579](19/19)[559-577](18/19) [440-458](19/19) [681-699](19/19) — chimpanzee 250human, chimpanzee UUGCGAGGUUGUGUUAUGC GCAUAACACAACCUCGCAA[510-528](19/19) [514-526](13/13) — [630-648](19/19) — 251 human, mouse,UUGCUCAAGAUGUCCUGCG CGCAGGACAUCUUGAGCAA [466-484](19/19)[464-482](19/19) [345-363](18/19) [586-604](19/19) [448-466]chimpanzee, dog (19/19) 252 human, mouse, ACCCAGAGAAUUGCUCAAGCUUGAGCAAUUCUCUGGGU [456-474](19/19) [454-472](19/19) [335-353](18/19)[576-594](19/19) — chimpanzee 253 human, chimpanzee UCAAAGCAAACUAAACUUGCAAGUUUAGUUUGCUUUGA [405-423](19/19) [403-421](18/19) [284-300](16/17)[525-543](19/19) [387-397] (11/11) 254 human, chimpanzeeUCCAAAUCAAAGCAAACUA UAGUUUGCUUUGAUUUGGA [399-417](19/19)[397-413](16/17) [278-294](16/17) [519-537](19/19) [381-397] (17/17) 255human AUGGAAGGCUGUUAAAUUA UAAUUUAACAGCCUUCCAU [2510-2528](19/19) — — — —256 human, chimpanzee, CUGUCCAAAUCAAAGCAAA UUUGCUUUGAUUUGGACAG[396-414](19/19) [394-412](18/19) — [516-534](19/19) [378-396] dog(19/19) 257 human, chimpanzee, GUCUGUCCAAAUCAAAGCA UGCUUUGAUUUGGACAGAC[394-412](19/19) — — [514-532](19/19) [376-394] dog (19/19) 258 humanGUUAUGCUUACAAAAUGGU ACCAUUUUGUAAGCAUAAC [2483-2501](19/19) — — — — 259human UUGACUCUCUUGCCUGUUA UAACAGGCAAGAGAGUCAA [2468-2486](19/19) — — — —260 human CCUUUUUCCACUAACAGUU AACUGUUAGUGGAAAAAGG [2445-2463](19/19) — —— — 261 human, chimpanzee, GAACUGUCUGUCCAAAUCA UGAUUUGGACAGACAGUUC[389-407](19/19) [387-405](18/19) [268-282](15/15) [509-527](19/19)[371-389] dog (19/19) 262 human UGGGCAUCGAUGUAGAACU AGUUCUACAUCGAUGCCCA[2421-2439](19/19) — — — — 263 human AAAGGUUCACUGUGUUUCUAGAAACACAGUGAACCUUU [2359-2377](19/19) — — — — 264 humanUCCAAAGGUUCACUGUGUU AACACAGUGAACCUUUGGA [2356-2374](19/19) — — — — 265human GCAUGUCUAUUGUUAAGCU AGCUUAACAAUAGACAUGC [2338-2356](19/19) — — — —266 human UCAAUGUUGUUUUGCAUGU ACAUGCAAAACAACAUUGA [2325-2343](19/19) — —— — 267 human UUGGGCCAAGAUAAAUCAA UUGAUUUAUCUUGGCCCAA [2310-2328](19/19)— — — — 268 human AUUGGGCCAAGAUAAAUCA UGAUUUAUCUUGGCCCAAU[2309-2327](19/19) — — — — 269 human CAAAGCCAAUCUUUAUAGAUCUAUAAAGAUUGGCUUUG [2290-2308](19/19) — — — — 270 humanCUACAAAGCCAAUCUUUAU AUAAAGAUUGGCUUUGUAG [2287-2305](19/19) — — — — 271human ACUAAACUCUUCAAAUGCU AGCAUUUGAAGAGUUUAGU [2258-2276](19/19) — — — —272 human GUGUUCUAACAAACUAAAC GUUUAGUUUGUUAGAACAC [2246-2264](19/19) — —— — 273 human UCACUGUUCAAAUUAGCCA UGGCUAAUUUGAACAGUGA [2227-2245](19/19)— — — — 274 human CUGUUACUGAUACUAUAAG CUUAUAGUAUCAGUAACAG[2196-2214](19/19) — — — — 275 human UCCUGUUACUGAUACUAUAUAUAGUAUCAGUAACAGGA [2194-2212](19/19) — — — — 276 humanAUCCUGUUACUGAUACUAU AUAGUAUCAGUAACAGGAU [2193-2211](19/19) — — — — 277human CAGGUGUGAUCCUGUUACU AGUAACAGGAUCACACCUG [2185-2203](19/19) — — — —278 human UAGGGACAGAUGUAUUCAU AUGAAUACAUCUGUCCCUA [2148-2166](19/19) — —— — 279 human GCUAUUAGCUCCACUUCAC GUGAAGUGGAGCUAAUAGC [2113-2131](19/19)— — — — 280 human GCCCUAGCUAUUAGCUCCA UGGAGCUAAUAGCUAGGGC[2107-2125](19/19) — — — — 281 human UCGUGGAUAAGGAGCUUAUAUAAGCUCCUUAUCCACGA [2077-2095](19/19) — — — — 282 humanAAGGCUUUCAUAUCCUUGC GCAAGGAUAUGAAAGCCUU [2052-2070](19/19) — — — — 283human CCACUCAACAAUGUUCAAU AUUGAACAUUGUUGAGUGG [2028-2046](19/19) — — — —284 human CCUCCACUCAACAAUGUUC GAACAUUGUUGAGUGGAGG [2025-2043](19/19) — —— — 285 human GGAUACCUGUCACUAGGGA UCCCUAGUGACAGGUAUCC [1923-1941](19/19)— — — — 286 human ACCUUGGAUACCUGUCACU AGUGACAGGUAUCCAAGGU[1918-1936](19/19) — — — — 287 human UCACCGUCCAGAUAACCAUAUGGUUAUCUGGACGGUGA [1878-1896](19/19) — — — — 288 humanAACUCACCGUCCAGAUAAC GUUAUCUGGACGGUGAGUU [1875-1893](19/19) — — — — 289human GAGAUAUGGUUUAUAGUAC GUACUAUAAACCAUAUCUC [1842-1860](19/19) — — — —290 human GCAUUGGCUAUGGAGAUAU AUAUCUCCAUAGCCAAUGC [1830-1848](19/19) — —— — 291 human, chimpanzee AACGAGGUAAUAUUUGAGG CCUCAAAUAUUACCUCGUU[336-354](19/19) — — [456-474](19/19) — 292 human, chimpanzeeCAACGAGGUAAUAUUUGAG CUCAAAUAUUACCUCGUUG [335-353](19/19) — —[455-473](19/19) — 293 human CUGUAUACUACCACUUUGA UCAAAGUGGUAGUAUACAG[1781-1799](19/19) — — — — 294 human UAGCUGUAUACUACCACUUAAGUGGUAGUAUACAGCUA [1778-1796](19/19) — — — — 295 humanGUAGCUGUAUACUACCACU AGUGGUAGUAUACAGCUAC [1777-1795](19/19) — — — — 296human UGGCAGUGUUAUCUCAUCU AGAUGAGAUAACACUGCCA [1704-1722](19/19) — — — —297 human UCUCAGAUGAACCAUUUCA UGAAAUGGUUCAUCUGAGA [1682-1700](19/19) — —— — 298 human UAAUCUCAGAUGAACCAUU AAUGGUUCAUCUGAGAUUA [1679-1697](19/19)— — — — 299 human CCCUCUCUGAUUCACUUAG CUAAGUGAAUCAGAGAGGG[1628-1646](19/19) — — — — 300 human AGUCCCUCUCUGAUUCACUAGUGAAUCAGAGAGGGACU [1625-1643](19/19) [1278-1288] — — — (11/11) 301human UAGUCCCUCUCUGAUUCAC GUGAAUCAGAGAGGGACUA [1624-1642](19/19)[1278-1288] — — — (11/11) 302 human UUGAUUACUCUUCCAUUGAUCAAUGGAAGAGUAAUGAA [1560-1578](19/19) — — — — 303 humanGUGUAGUUGAUUACUCUUC GAAGAGUAAUCAACUACAC [1554-1572](19/19) — — — — 304human CCCCUGUUCUUAAGUGUUG CAACACUUAAGAACAGGGG [1484-1502](19/19) — — — —305 human UUCCCCUGUUCUUAAGUGU ACACUUAAGAACAGGGGAA [1482-1500](19/19) — —— — 306 human CAGUUUCCCCUGUUCUUAA UUAAGAACAGGGGAAACUG [1478-1496](19/19)— — — — 307 human GCCUUUAUAAGCUCAGUUU AAACUGAGCUUAUAAAGGC[1465-1483](19/19) — — — — 308 human GUGUUGUUUUAGAUGCCUUAAGGCAUCUAAAACAACAC [1451-1469](19/19) — — — — 309 humanACGGUGUUGUUUUAGAUGC GCAUCUAAAACAACACCGU [1448-1466](19/19) — — — — 310human CCACGGUGUUGUUUUAGAU AUCUAAAACAACACCGUGG [1446-1464](19/19) — — — —311 human UCAGUAACCACGGUGUUGU ACAACACCGUGGUUACUGA [1439-1457](19/19) — —— — 312 human CCAUUUCAGUAACCACGGU ACCGUGGUUACUGAAAUGG [1434-1452](19/19)— — — — 313 human GUAGGAUUAAGUAGGUGAG CUCACCUACUUAAUCCUAC[1289-1307](19/19) — — — — 314 human GGUAGGAUUAAGUAGGUGAUCACCUACUUAAUCCUACC [1288-1306](19/19) — — — — 315 humanAAGGGUUUUUAGACAGGAA UUCCUGUCUAAAAACCCUU [1269-1287](19/19) — — — — 316human UUGAAGGGUUUUUAGACAG CUGUCUAAAAACCCUUCAA [1266-1284](19/19) — — — —317 human CAGUUCCUGACUCAAAUUU AAAUUUGAGUCAGGAACUG [1249-1267](19/19) — —— — 318 human ACCAGUUCCUGACUCAAAU AUUUGAGUCAGGAACUGGU [1247-1265](19/19)— — — — 319 human CCUAUCAAAACUUCCAAAA UUUUGGAAGUUUUGAUAGG[1219-1237](19/19) — — — [1182-1194] (13/13) 320 human, chimpanzeeUGCUAAGUGAUUUUGACUA UAGUCAAAAUCACUUAGCA [286-304](19/19)[284-302](18/19) [165-183](18/19) [406-424](19/19) [268-286] (18/19) 321human UCCACAAUUUGGUUUCAGG CCUGAAACCAAAUUGUGGA [1081-1099](19/19) — — — —322 human UGAUUCCACAAUUUGGUUU AAACCAAAUUGUGGAAUCA [1077-1095](19/19) — —— — 323 human AUAGAUCCCAUUUUUGUAC GUACAAAAAUGGGAUCUAU [1000-1018](19/19)— — — — 324 human, chimpanzee CAGAGAGCCUGCUAAGUGA UCACUUAGCAGGCUCUCUG[277-295](19/19) [283-293](11/11) [164-174](11/11) [397-415](19/19)[263-273] (11/11) 325 human GUAGAUUUUCUGCAUAGAU AUCUAUGCAGAAAAUCUAC[987-1005](19/19) — — — — 326 human UAGUGUAGAUUUUCUGCAUAUGCAGAAAAUCUACACUA [983-1001](19/19) — — — — 327 humanAUAGUGUAGAUUUUCUGCA UGCAGAAAAUCUACACUAU [982-1000](19/19) — — — — 328human UGUUUCACAUUCAUAGCAA UUGCUAUGAAUGUGAAACA [916-934](19/19) — — — —329 human CACCUGCCCUAAAUAAGAA UUCUUAUUUAGGGCAGGUG [869-887](19/19) — — —— 330 human AAAGUCAUUUGUAGUUUGC GCAAACUACAAAUGACUUU [848-866](19/19)[845-861](17/17) — — [834-845] (12/12) 331 human GCUAGUCAGCUAAAGUCAUAUGACUUUAGCUGACUAGC [837-855](19/19) — — — — 332 humanUCAGCUAGUCAGCUAAAGU ACUUUAGCUGACUAGCUGA [834-852](19/19) — — — — 333human GGGUAGUAAAACUAUUCAG CUGAAUAGUUUUACUACCC [819-837](19/19) — — — —334 human, chimpanzee CCAGCAUUUCAGAAUUGCU AGCAAUUCUGAAAUGCUGG[241-259](19/19) [239-251](13/13) [120-138](18/19) [361-379](19/19) —335 human, mouse CAGCUCAGGAUUUCGACUU AAGUCGAAAUCCUGAGCUG[713-731](19/19) [711-729](19/19) [592-610](18/19) — — 336 human, ratGAACUCUGAUCCUCAGCUC GAGCUGAGGAUCAGAGUUC [700-718](19/19) —[579-597](19/19) — — 337 human CGCUUCUCCUCUGGUUUCA UGAAACCAGAGGAGAAGCG[678-696](19/19) [676-686](11/11) — — — 338 human GACUUUUUCUUUAGUAGAGCUCUACUAAAGAAAAAGUC [657-675](19/19) — — [777-789](13/13) — 339 humanUCAGGGACUUUUUCUUUAG CUAAAGAAAAAGUCCCUGA [652-670](19/19) — —[772-789](18/18) — 340 human, chimpanzee UUCAGGGACUUUUUCUUUAUAAAGAAAAAGUCCCUGAA [651-669](19/19) — — [771-789](19/19) — 341human, chimpanzee CUUUUGAGCUUACACUUGU ACAAGUGUAAGCUCAAAAG[601-619](19/19) — — [721-739](19/19) — 342 human UACCUACUUUUGAGCUUACGUAAGCUCAAAAGUAGGUA [595-613](19/19) — — [717-733](17/17) — 343 humanGUACCUACUUUUGAGCUUA UAAGCUCAAAAGUAGGUAC [594-612](19/19) — —[717-732](16/16) — 344 human, rat, GUGAACUUGGAAAUUGAAAUUUCAAUUUCCAAGUUCAC [531-549](19/19) [529-547](18/19) [410-428](19/19)[651-669](19/19) [513-531] chimpanzee, dog (19/19) 345 human, rat, dogGCACGUGAACUUGGAAAUU AAUUUCCAAGUUCACGUGC [527-545](19/19)[525-539](15/15) [406-424](19/19) [651-665](15/15) [509-527] (19/19) 346human, mouse, UCCCUGAGAAACUGACCCA UGGGUCAGUUUCUCAGGGA [442-460](19/19)[440-458](19/19) [323-339](17/17) [562-580](19/19) [424-442]chimpanzee, dog (19/19) 347 human, chimpanzee AGGUCCUUGUCCCUGAGAAUUCUCAGGGACAAGGACCU [433-451](19/19) [439-449](11/11) — [553-571](19/19)[417-433] (17/17) 348 human, chimpanzee GCAAACUAAACUUGGUUGCGCAACCAAGUUUAGUUUGC [410-428](19/19) [408-426](18/19) — [530-548](19/19)— 349 human, chimpanzee GUCCAAAUCAAAGCAAACU AGUUUGCUUUGAUUUGGAC[398-416](19/19) [396-413](17/18) [277-294](17/18) [518-536](19/19)[380-397] (18/18) 350 human, chimpanzee, UCUGUCCAAAUCAAAGCAAUUGCUUUGAUUUGGACAGA [395-413](19/19) — — [515-533](19/19) [377-395] dog(19/19) 351 human CCUGUUAUGCUUACAAAAU AUUUUGUAAGCAUAACAGG[2480-2498](19/19) — — — — 352 human GCCUGUUAUGCUUACAAAAUUUUGUAAGCAUAACAGGC [2479-2497](19/19) — — — — 353 humanCAGUUAUCUUUGACUCUCU AGAGAGUCAAAGAUAACUG [2459-2477](19/19) — — — — 354human CACUAACAGUUAUCUUUGA UCAAAGAUAACUGUUAGUG [2453-2471](19/19) — — — —355 human UCCACUAACAGUUAUCUUU AAAGAUAACUGUUAGUGGA [2451-2469](19/19) — —— — 356 human, chimpanzee, AGAACUGUCUGUCCAAAUC GAUUUGGACAGACAGUUCU[388-406](19/19) [386-400](15/15) [267-282](16/16) [508-526](19/19)[370-388] dog (19/19) 357 human UGACUGGGCAAGGCUUCUU AAGAAGCCUUGCCCAGUCA[2403-2421](19/19) — — — — 358 human UCACUGUGUUUCUGCCGCUAGCGGCAGAAACACAGUGA [2365-2383](19/19) [2460-2470] — — — (11/11) 359human AAGGUUCACUGUGUUUCUG CAGAAACACAGUGAACCUU [2360-2378](19/19)[2460-2470] — — — (11/11) 360 human CCAAGAUAAAUCAAUGUUGCAACAUUGAUUUAUCUUGG [2315-2333](19/19) — — — — 361 humanGAUACUACAAAGCCAAUCU AGAUUGGCUUUGUAGUAUC [2283-2301](19/19) [1571-1581] —— — (11/11) 362 human AAAGAUACUACAAAGCCAA UUGGCUUUGUAGUAUCUUU[2280-2298](19/19) [1571-1581] — — — (11/11) 363 humanGAAAGAUACUACAAAGCCA UGGCUUUGUAGUAUCUUUC [2279-2297](19/19) [1571-1581] —— — (11/11) 364 human UGGAAAGAUACUACAAAGC GCUUUGUAGUAUCUUUCCA[2277-2295](19/19) [1571-1581] — — — (11/11) 365 humanUUGGAAAGAUACUACAAAG CUUUGUAGUAUCUUUCCAA [2276-2294](19/19) [1571-1581] —— — (11/11) 366 human UGCUUGGAAAGAUACUACA UGUAGUAUCUUUCCAAGCA[2273-2291](19/19) — — — — 367 human AUGCUUGGAAAGAUACUACGUAGUAUCUUUCCAAGCAU [2272-2290](19/19) — — — — 368 humanCUAAACUCUUCAAAUGCUU AAGCAUUUGAAGAGUUUAG [2259-2277](19/19) — — — — 369human AAGUGACCUAAAAUGUCAC GUGACAUUUUAGGUCACUU [2212-2230](19/19) — — — —370 human UGAUCCUGUUACUGAUACU AGUAUCAGUAACAGGAUCA [2191-2209](19/19) — —— — 371 human ACAGGUGUGAUCCUGUUAC GUAACAGGAUCACACCUGU [2184-2202](19/19)— — — — 372 human AUCCUGGUGUUACUGAAAA UUUUCAGUAACACCAGGAU[2165-2183](19/19) — — — — 373 human GGACAGAUGUAUUCAUCCUAGGAUGAAUACAUCUGUCC [2151-2169](19/19) — — — — 374 humanAGCUCCACUUCACAUGCUG CAGCAUGUGAAGUGGAGCU [2119-2137](19/19) — — — — 375human AGCUAUUAGCUCCACUUCA UGAAGUGGAGCUAAUAGCU [2112-2130](19/19) — — — —376 human, chimpanzee CUUGCCAGAAUUUGGUUAA UUAACCAAAUUCUGGCAAG[361-379](19/19)  [361-377](17/17) [242-258](17/17) [481-499](19/19) —377 human AAGGAGCUUAUUCAGGUUU AAACCUGAAUAAGCUCCUU [2085-2103](19/19) — —— — 378 human GCUUUCAUAUCCUUGCUGU ACAGCAAGGAUAUGAAAGC [2055-2073](19/19)— — — — 379 human UGAGAGAAUUUAGCCUCCA UGGAGGCUAAAUUCUCUCA[2012-2030](19/19) — — — — 380 human AUGAGAGAAUUUAGCCUCCGGAGGCUAAAUUCUCUCAU [2011-2029](19/19) — — — — 381 human, chimpanzeeGGAAUCAACUUGCCAGAAU AUUCUGGCAAGUUGAUUCC [353-371](19/19) — —[473-491](19/19) [340-351] (12/12) 382 human UAGGGAAUAAUAAAGGCCUAGGCCUUUAUUAUUCCCUA [1936-1954](19/19) — — — — 383 humanUCACUAGGGAAUAAUAAAG CUUUAUUAUUCCCUAGUGA [1932-1950](19/19) — — — — 384human ACCUGUCACUAGGGAAUAA UUAUUCCCUAGUGACAGGU [1927-1945](19/19) — — — —385 human AUACCUGUCACUAGGGAAU AUUCCCUAGUGACAGGUAU [1925-1943](19/19) — —— — 388 human UUGGAUACCUGUCACUAGG CCUAGUGACAGGUAUCCAA [1921-1939](19/19)— — — — 387 human CACCUUGGAUACCUGUCAC GUGACAGGUAUCCAAGGUG[1917-1935](19/19) — — — — 388 human ACCGUCCAGAUAACCAUGCGCAUGGUUAUCUGGACGGU [1880-1898](19/19) — — — — 389 humanGAAGUCUGCAUUGGCUAUG CAUAGCCAAUGCAGACUUC [1823-1841](19/19) — — — — 390human GAAUUAUUGAAACGGGUCA UGACCCGUUUCAAUAAUUC [1798-1816](19/19) — — — —391 human UAGUAGCUGUAUACUACCA UGGUAGUAUACAGCUACUA [1775-1793](19/19) — —— — 392 human GGGUAGUAGCUGUAUACUA UAGUAUACAGCUACUACCC [1772-1790](19/19)— — — — 393 human GUCAAGGGUAGUAGCUGUA UACAGCUACUACCCUUGAC[1767-1785](19/19) — — — — 394 human UGAAGUAUCUCUCCUUAACGUUAAGGAGAGAUACUUCA [1741-1759](19/19) — — — — 395 humanUUGAAGUAUCUCUCCUUAA UUAAGGAGAGAUACUUCAA [1740-1758](19/19) — — — — 396human GGGAAUUGAAGUAUCUCUC GAGAGAUACUUCAAUUCCC [1735-1753](19/19) — — — —397 human UGGGAAUUGAAGUAUCUCU AGAGAUACUUCAAUUCCCA [1734-1752](19/19) — —— — 398 human GGCUUUUCUGGGAAUUGAA UUCAAUUCCCAGAAAAGCC [1726-1744](19/19)— — — — 399 human, chimpanzee CCCAACCUCAACGAGGUAA UUACCUCGUUGAGGUUGGG[327-345](19/19) [325-341](17/17) [206-222](17/17) [447-465](19/19)[309-325] (16/17) 400 human GCAGUGUUAUCUCAUCUCU AGAGAUGAGAUAACACUGC[1706-1724](19/19) — — — — 401 human CUCAGAUGAACCAUUUCACGUGAAAUGGUUCAUCUGAG [1683-1701](19/19) — — — — 402 human, chimpanzeeCUGAACCCAACCUCAACGA UCGUUGAGGUUGGGUUCAG [322-340](19/19)[320-338](18/19) [206-219](14/14) [442-460](19/19) [306-319] (14/14) 403human CUGAUUCACUUAGUAAUCU AGAUUACUAAGUGAAUCAG [1634-1652](19/19) — — — —404 human CUCUGAUUCACUUAGUAAU AUUACUAAGUGAAUCAGAG [1632-1650](19/19) — —— — 405 human CCUCUCUGAUUCACUUAGU ACUAAGUGAAUCAGAGAGG [1629-1647](19/19)— — — — 406 human, chimpanzee AGGAAACAGAGCCGUUGAC GUCAACGGCUCUGUUUCCU[184-202](19/19) [183-200](18/18) [64-81](18/18) [304-322](19/19)[167-184] (18/18) 407 human UACUCUUCCAUUGAGUGAA UUCACUCAAUGGAAGAGUA[1565-1583](19/19) — — — — 408 human GUGUGUAGUUGAUUACUCUAGAGUAAUCAACUACACAC [1552-1570](19/19) — — — — 409 humanGAACUGAUAUUUUUGUGUG CACACAAAAAUAUCAGUUC [1538-1556](19/19) — — — — 410human AAGUGUUGAAUACUGUCUU AAGACAGUAUUCAACACUU [1495-1513](19/19) — — — —411 human UCCCCUGUUCUUAAGUGUU AACACUUAAGAACAGGGGA [1483-1501](19/19) — —— — 412 human GCUCAGUUUCCCCUGUUCU AGAACAGGGGAAACUGAGC [1475-1493](19/19)— — — — 413 human AGCUCAGUUUCCCCUGUUC GAACAGGGGAAACUGAGCU[1474-1492](19/19) — — — — 414 human, chimpanzee GGAUUAUGUUGUUCCUGAAUUCAGGAACAACAUAAUCC [308-326](19/19) [306-323](17/18) — [428-446](19/19)— 415 human UAAGCUCAGUUUCCCCUGU ACAGGGGAAACUGAGCUUA [1472-1490](19/19) —— — — 416 human UGCCUUUAUAAGCUCAGUU AACUGAGCUUAUAAAGGCA[1464-1482](19/19) — — — — 417 human AGAUGCCUUUAUAAGCUCAUGAGCUUAUAAAGGCAUCU [1461-1479](19/19) — — — — 418 humanUUAGAUGCCUUUAUAAGCU AGCUUAUAAAGGCAUCUAA [1459-1477](19/19) — — — — 419human CGGUGUUGUUUUAGAUGCC GGCAUCUAAAACAACACCG [1449-1467](19/19) — — — —420 human UAACCACGGUGUUGUUUUA UAAAACAACACCGUGGUUA [1443-1461](19/19) — —— — 421 human, chimpanzee CUACUGGGAUUAUGUUGUU AACAACAUAAUCCCAGUAG[302-320](19/19) [300-317](18/18) [181-195](15/15) [422-440](19/19)[284-302] (18/19) 422 human, mouse, ACUACUGGGAUUAUGUUGUACAACAUAAUCCCAGUAGU [301-319](19/19) [299-317](19/19) [180-195](16/16)[421-439](19/19) [283-301] chimpanzee (18/19) 423 humanAUGGAACUGCUUCACUGUU AACAGUGAAGCAGUUCCAU [1361-1379](19/19) — — — — 424human UUACGGCAAUAAUGGAACU AGUUCCAUUAUUGCCGUAA [1350-1368](19/19) — — — —425 human AGGUGAGUUUAAUUAAAGC GCUUUAAUUAAACUCACCU [1301-1319](19/19) — —— — 426 human AGGAUUAAGUAGGUGAGUU AACUCACCUACUUAAUCCU [1291-1309](19/19)— — — — 427 human ACAGGAAGGUAGGAUUAAG CUUAAUCCUACCUUCCUGU[1281-1299](19/19) — — — — 428 human AGGGUUUUUAGACAGGAAGCUUCCUGUCUAAAAACCCU [1270-1288](19/19) — — — — 429 humanGAAGGGUUUUUAGACAGGA UCCUGUCUAAAAACCCUUC [1268-1286](19/19) — — — — 430human UGAAGGGUUUUUAGACAGG CCUGUCUAAAAACCCUUCA [1267-1285](19/19) — — — —431 human UUUGAAGGGUUUUUAGACA UGUCUAAAAACCCUUCAAA [1265-1283](19/19) — —— — 432 human, chimpanzee GUGAUUUUGACUACUGGGA UCCCAGUAGUCAAAAUCAC[292-310](19/19) [290-308](18/19) [171-189](18/19) [412-430](19/19)[275-292] (18/18) 433 human UCCUGACUCAAAUUUGAAG CUUCAAAUUUGAGUCAGGA[1253-1271](19/19) — — — — 434 human UCUCUAAGUUUUCAGAGGAUCCUCUGAAAACUUAGAGA [1163-1181](19/19) — — — — 435 humanAGGUAGGCUUGGUAAUAGA UCUAUUACCAAGCCUACCU [1097-1115](19/19) — — — — 436human ACAAUUUGGUUUCAGGUAG CUACCUGAAACCAAAUUGU [1084-1102](19/19) — — — —437 human GUUUUACAUUUGAUUCCAC GUGGAAUCAAAUGUAAAAC [1067-1085](19/19) — —— — 438 human AAGCCCAUUUGAGUUUUAC GUAAAACUCAAAUGGGCUU [1055-1073](19/19)— — — — 439 human UAGAAGCCCAUUUGAGUUU AAACUCAAAUGGGCUUCUA[1052-1070](19/19) — — — — 440 human AAGCAAAAGUAGAAGCCCAUGGGCUUCUACUUUUGCUU [1043-1061](19/19) — — — — 441 humanUAGGUAAGCAAAAGUAGAA UUCUACUUUUGCUUACCUA [1038-1056](19/19) — — — — 442human GAAUAGGUAAGCAAAAGUA UACUUUUGCUUACCUAUUC [1035-1053](19/19) — — — —443 human GGAUGGAAUAGGUAAGCAA UUGCUUACCUAUUCCAUCC [1030-1048](19/19) — —— — 444 human GGGAUGGAAUAGGUAAGCA UGCUUACCUAUUCCAUCCC [1029-1047](19/19)— — — — 445 human AGAAUUGAAUGGGAUGGAA UUCCAUCCCAUUCAAUUCU[1019-1037](19/19) — — — — 446 human CUGCAUAGAUCCCAUUUUUAAAAAUGGGAUCUAUGCAG [996-1014](19/19) — — — — 447 humanUAGAUUUUCUGCAUAGAUC GAUCUAUGCAGAAAAUCUA [988-1006](19/19) — — — — 448human GGCCUUUGGAGAAGUGAUU AAUCACUUCUCCAAAGGCC [959-977](19/19) — — — —449 human, chimpanzee ACUGUGGCUAUCACCCAGA UCUGGGUGAUAGCCACAGU[262-280](19/19) [263-275](13/13) — [382-400](19/19) — 450 humanGGCUGAUUUUCUGGCCUUU AAAGGCCAGAAAAUCAGCC [947-965](19/19) — — — — 451human ACAGGCUGAUUUUCUGGCC GGCCAGAAAAUCAGCCUGU [944-962](19/19)[939-949](11/11) — — — 452 human AACAGGCUGAUUUUCUGGC GCCAGAAAAUCAGCCUGUU[943-961](19/19) [939-949](11/11) — — — 453 human CUGCAGCUAACAGGCUGAUAUCAGCCUGUUAGCUGCAG [935-953](19/19) — — — — 454 humanAGCAACUGCAGCUAACAGG CCUGUUAGCUGCAGUUGCU [930-948](19/19) — — — — 455human UAGCAACUGCAGCUAACAG CUGUUAGCUGCAGUUGCUA [929-947](19/19) — — — —456 human UCAUAGCAACUGCAGCUAA UUAGCUGCAGUUGCUAUGA [926-944](19/19) — — —— 457 human UCACAUUCAUAGCAACUGC GCAGUUGCUAUGAAUGUGA [920-938](19/19) — —— — 458 human, chimpanzee CAGAAUUGCUGGACUGUGG CCACAGUCCAGCAAUUCUG[250-268](19/19) — — [370-388](19/19) — 459 human GCCCUAAAUAAGAAACCCCGGGGUUUCUUAUUUAGGGC [874-892](19/19) — — — — 460 humanCCACCUGCCCUAAAUAAGA UCUUAUUUAGGGCAGGUGG [868-886](19/19) — — — — 461human GUCAUUUGUAGUUUGCCCC GGGGCAAACUACAAAUGAC [851-869](19/19)[846-862](17/17) — — [834-845] (12/12) 462 human GUAAAACUAUUCAGCUAGUACUAGCUGAAUAGUUUUAC [824-842](19/19) — — — — 463 humanUUGGGUAGUAAAACUAUUC GAAUAGUUUUACUACCCAA [817-835](19/19) — — — — 464human CCUCAGCUCAGGAUUUCGA UCGAAAUCCUGAGCUGAGG [710-728](19/19)[709-726](18/18) — — — 465 human GGAGAACUCUGAUCCUCAG CUGAGGAUCAGAGUUCUCC[697-715](19/19) — [579-594](16/16) — — 466 human UCGCUUCUCCUCUGGUUUCGAAACCAGAGGAGAAGCGA [677-695](19/19) [675-686](12/12) — — — 467 humanGUCGCUUCUCCUCUGGUUU AAACCAGAGGAGAAGCGAC [676-694](19/19)[674-686](13/13) — — — 468 human GGACUUUUUCUUUAGUAGA UCUACUAAAGAAAAAGUCC[656-674](19/19) — — [776-789](14/14) — 469 human, chimpanzeeUGGACUAGCUUCAGGGACU AGUCCCUGAAGCUAGUCCA [642-660](19/19) — —[762-780](19/19) [624-640] (17/17) 470 human, chimpanzee,UGCUCAUGGACUAGCUUCA UGAAGCUAGUCCAUGAGCA [636-654](19/19) — —[756-774](19/19) [618-636] dog (19/19) 471 human ACCUACUUUUGAGCUUACAUGUAAGCUCAAAAGUAGGU [596-614](19/19) — — [717-734](18/18) — 472 humanUCGUACCUACUUUUGAGCU AGCUCAAAAGUAGGUACGA [592-610](19/19) — —[712-730](18/19) — 473 human CGUCGUACCUACUUUUGAG CUCAAAAGUAGGUACGACG[590-608](19/19) — — [711-728](17/18) — 474 human, rat, dogCACGUGAACUUGGAAAUUG CAAUUUCCAAGUUCACGUG [528-546](19/19)[526-544](18/19) [407-425](19/19) [651-666](16/16) [510-528] (19/19) 475human, chimpanzee UGCGAGGUUGUGUUAUGCA UGCAUAACACAACCUCGCA[511-529](19/19) [514-527](14/14) [398-408](11/11) [631-649](19/19)[493-511] (18/19) 476 human, chimpanzee, UUGUCCCUGAGAAACUGACGUCAGUUUCUCAGGGACAA [439-457](19/19) [439-455](17/17) [323-336](14/14)[559-577](19/19) [421-439] dog (19/19) 477 human, chimpanzee,UCCUUGUCCCUGAGAAACU AGUUUCUCAGGGACAAGGA [436-454](19/19)[439-452](14/14) [323-333](11/11) [556-574](19/19) [418-436] dog (19/19)478 human, chimpanzee UGACCAUGGUUGCAACUGG CCAGUUGCAACCAUGGUCA[199-217](19/19) [197-212](16/16) [78-93](16/16) [319-337](19/19) — 479human, chimpanzee AACUAAACUUGGUUGCUCA UGAGCAACCAAGUUUAGUU[413-431](19/19) [415-428](14/14) — [533-551](19/19) [402-413] (12/12)480 human, chimpanzee AGCAAACUAAACUUGGUUG CAACCAAGUUUAGUUUGCU[409-427](19/19) [407-425](18/19) — [529-547](19/19) — 481 humanUGUUAUGCUUACAAAAUGG CCAUUUUGUAAGCAUAACA [2482-2500](19/19) — — — — 482human, chimpanzee, ACUGUCUGUCCAAAUCAAA UUUGAUUUGGACAGACAGU[391-409](19/19) [389-407](18/19) [270-288](18/19) [511-529](19/19)[373-391] dog (19/19) 483 human CUAACAGUUAUCUUUGACU AGUCAAAGAUAACUGUUAG[2455-2473](19/19) — — — — 484 human GGGCAUCGAUGUAGAACUGCAGUUCUACAUCGAUGCCC [2422-2440](19/19) — — — — 485 humanUCCUGGAGUUGUCACCACU AGUGGUGACAACUCCAGGA [2385-2403](19/19) — — — — 486human UUGUUAAGCUCCAAAGGUU AACCUUUGGAGCUUAACAA [2347-2365](19/19) — — — —487 human UUGCAUGUCUAUUGUUAAG CUUAACAAUAGACAUGCAA [2336-2354](19/19) — —— — 488 human AUGUUGUUUUGCAUGUCUA UAGACAUGCAAAACAACAU [2328-2346](19/19)— — — — 489 human AAGAUACUACAAAGCCAAU AUUGGCUUUGUAGUAUCUU[2281-2299](19/19) [1571-1581] — — — (11/11) 490 humanCUUGGAAAGAUACUACAAA UUUGUAGUAUCUUUCCAAG [2275-2293](19/19) — — — — 491human UGUCACUGUUCAAAUUAGC GCUAAUUUGAACAGUGACA [2225-2243](19/19) — — — —492 human UGAUACUAUAAGUGACCUA UAGGUCACUUAUAGUAUCA [2203-2221](19/19) — —— — 493 human, mouse, CCGUUGACCAUGGUUGCAA UUGCAACCAUGGUCAACGG[195-213](19/19) [193-211](19/19) [74-92](19/19) [315-333](19/19)[177-195] rat, chimpanzee (18/19) 494 human GUGUUACUGAAAAACAGGUACCUGUUUUUCAGUAACAC [2171-2189](19/19) — — — — 495 humanAGGGACAGAUGUAUUCAUC GAUGAAUACAUCUGUCCCU [2149-2167](19/19) — — — — 496human GGCGUAGGGACAGAUGUAU AUACAUCUGUCCCUACGCC [2144-2162](19/19) — — — —497 human CUCCACUUCACAUGCUGGA UCCAGCAUGUGAAGUGGAG [2121-2139](19/19) — —— — 498 human UUCCUGCCCUAGCUAUUAG CUAAUAGCUAGGGCAGGAA [2102-2120](19/19)— — — — 499 human AGGAGCUUAUUCAGGUUUC GAAACCUGAAUAAGCUCCU[2086-2104](19/19) — — — — 500 human AUAAGGAGCUUAUUCAGGUACCUGAAUAAGCUCCUUAU [2083-2101](19/19) — — — — 501 human, chimpanzeeCAACUUGCCAGAAUUUGGU ACCAAAUUCUGGCAAGUUG [358-376](19/19)[356-374](18/19) [237-255](18/19) [478-496](19/19) [340-357] (17/18) 502human AAUUCAGCAAGGCUUUCAU AUGAAAGCCUUGCUGAAUU [2044-2062](19/19)[2157-2167] — — — (11/11) 503 human UCAACAAUGUUCAAUUCAGCUGAAUUGAACAUUGUUGA [2032-2050](19/19) — — — — 504 humanUCCACUCAACAAUGUUCAA UUGAACAUUGUUGAGUGGA [2027-2045](19/19) — — — — 505human UUCCAACUAAGUAGAUCAU AUGAUCUACUUAGUUGGAA [1969-1987](19/19) — — — —506 human AGGGAAUAAUAAAGGCCUU AAGGCCUUUAUUAUUCCCU [1937-1955](19/19) — —— — 507 human CACUAGGGAAUAAUAAAGG CCUUUAUUAUUCCCUAGUG [1933-1951](19/19)— — — — 508 human, chimpanzee GAGGAAUCAACUUGCCAGA UCUGGCAAGUUGAUUCCUC[351-369](19/19) — — [471-489](19/19) [340-351] (12/12) 509 humanCCUGUCACUAGGGAAUAAU AUUAUUCCCUAGUGACAGG [1928-1946](19/19) — — — — 510human CAUGCAUGCACCCAGAUUU AAAUCUGGGUGCAUGCAUG [1894-1912](19/19) — — — —511 human GUCCAGAUAACCAUGCAUG CAUGCAUGGUUAUCUGGAC [1883-1901](19/19) — —— — 512 human CCGUCCAGAUAACCAUGCA UGCAUGGUUAUCUGGACGG [1881-1899](19/19)— — — — 513 human GAAUGAAACUCACCGUCCA UGGACGGUGAGUUUCAUUC[1869-1887](19/19) — — — — 514 human GAGAAUGAAACUCACCGUCGACGGUGAGUUUCAUUCUC [1867-1885](19/19) — — — — 515 humanACAGCCUAGAGAAUGAAAC GUUUCAUUCUCUAGGCUGU [1859-1877](19/19) — — — — 516human CUGCAUUGGCUAUGGAGAU AUCUCCAUAGCCAAUGCAG [1828-1846](19/19) — — — —517 human AGUCUGCAUUGGCUAUGGA UCCAUAGCCAAUGCAGACU [1825-1843](19/19) — —— — 518 human AAGUCUGCAUUGGCUAUGG CCAUAGCCAAUGCAGACUU [1824-1842](19/19)— — — — 519 human UUGAAUUAUUGAAACGGGU ACCCGUUUCAAUAAUUCAA[1796-1814](19/19) — — — — 520 human GCUGUAUACUACCACUUUGCAAAGUGGUAGUAUACAGC [1780-1798](19/19) — — — — 521 humanAAGGGUAGUAGCUGUAUAC GUAUACAGCUACUACCCUU [1770-1788](19/19) — — — — 522human GAAGUAUCUCUCCUUAACC GGUUAAGGAGAGAUACUUC [1742-1760](19/19) — — — —523 human UUCUGGGAAUUGAAGUAUC GAUACUUCAAUUCCCAGAA [1731-1749](19/19) — —— — 524 human, chimpanzee AACCCAACCUCAACGAGGU ACCUCGUUGAGGUUGGGUU[325-343](19/19) [325-341](17/17) [206-222](17/17) [445-463](19/19)[307-325] (18/19) 525 human AUGAACCAUUUCACCAUGG CCAUGGUGAAAUGGUUCAU[1688-1706](19/19) — — — — 526 human AGAUGAACCAUUUCACCAUAUGGUGAAAUGGUUCAUCU [1686-1704](19/19) — — — — 527 humanAAUCUCAGAUGAACCAUUU AAAUGGUUCAUCUGAGAUU [1680-1698](19/19) — — — — 528human UGUGCUUAAUCUCAGAUGA UCAUCUGAGAUUAAGCACA [1673-1691](19/19) — — — —529 human AUGUGCUUAAUCUCAGAUG CAUCUGAGAUUAAGCACAU [1672-1690](19/19) — —— — 530 human ACAUGUGCUUAAUCUCAGA UCUGAGAUUAAGCACAUGU [1670-1688](19/19)— — — — 531 human UACAUGUGCUUAAUCUCAG CUGAGAUUAAGCACAUGUA[1669-1687](19/19) — — — — 532 human CCUCUUUUCAGUAUUACAUAUGUAAUACUGAAAAGAGG [1655-1673](19/19) — — — — 533 humanCUCUCUGAUUCACUUAGUA UACUAAGUGAAUCAGAGAG [1630-1648](19/19) — — — — 534human, chimpanzee AUGUUGUUCCUGAACCCAA UUGGGUUCAGGAACAACAU[313-331](19/19) — — [433-451](19/19) — 535 human CCCUGUUCUUAAGUGUUGAUCAACACUUAAGAACAGGG [1485-1503](19/19) — — — — 536 humanAUGCCUUUAUAAGCUCAGU ACUGAGCUUAUAAAGGCAU [1463-1481](19/19) — — — — 537human, chimpanzee CUGGGAUUAUGUUGUUCCU AGGAACAACAUAAUCCCAG[305-323](19/19) [303-317](15/15) [184-195](12/12) [425-443](19/19) —538 human CUAAUGUUUUAAAGAGGCA UGCCUCUUUAAAACAUUAG [1399-1417](19/19) — —— — 539 human, chimpanzee UUGACUACUGGGAUUAUGU ACAUAAUCCCAGUAGUCAA[298-316](19/19) [298-314](17/17) [179-195](17/17) [418-436](19/19)[280-295] (16/16) 540 human UGAGAAAUAUUACGGCAAU AUUGCCGUAAUAUUUCUCA[1341-1359](19/19) — — — — 541 human AUGAGAAAUAUUACGGCAAUUGCCGUAAUAUUUCUCAU [1340-1358](19/19) — — — — 542 humanGGUAAGAGUAAAUGAGAAA UUUCUCAUUUACUCUUACC [1329-1347](19/19) — — — — 543human CUAGGUAAGAGUAAAUGAG CUCAUUUACUCUUACCUAG [1326-1344](19/19) — — — —544 human AACCCUAGGUAAGAGUAAA UUUACUCUUACCUAGGGUU [1322-1340](19/19) — —— — 545 human GGUGAGUUUAAUUAAAGCU AGCUUUAAUUAAACUCACC [1302-1320](19/19)— — — — 546 human UAGACAGGAAGGUAGGAUU AAUCCUACCUUCCUGUCUA[1278-1296](19/19) — — — — 547 human UGACUCAAAUUUGAAGGGUACCCUUCAAAUUUGAGUCA [1256-1274](19/19) — — — — 548 humanCUGACUCAAAUUUGAAGGG CCCUUCAAAUUUGAGUCAG [1255-1273](19/19) — — — — 549human CACCAGUUCCUGACUCAAA UUUGAGUCAGGAACUGGUG [1246-1264](19/19) — — — —550 human ACCACCAGUUCCUGACUCA UGAGUCAGGAACUGGUGGU [1244-1262](19/19) — —— — 551 human AAAGCCCACACCACCAGUU AACUGGUGGUGUGGGCUUU [1235-1253](19/19)— — — — 552 human UAGGCUUGGUAAUAGACUA UAGUCUAUUACCAAGCCUA[1100-1118](19/19) — — — — 553 human CAAUUUGGUUUCAGGUAGGCCUACCUGAAACCAAAUUG [1085-1103](19/19) — — — — 554 humanAGCCCAUUUGAGUUUUACA UGUAAAACUCAAAUGGGCU [1056-1074](19/19) — — — — 555human, chimpanzee GCCUGCUAAGUGAUUUUGA UCAAAAUCACUUAGCAGGC[283-301](19/19) [283-296](14/14) [164-177](14/14) [403-421](19/19) —556 human AGCAAAAGUAGAAGCCCAU AUGGGCUUCUACUUUUGCU [1044-1062](19/19) — —— — 557 human GUAAGCAAAAGUAGAAGCC GGCUUCUACUUUUGCUUAC [1041-1059](19/19)— — — — 558 human, chimpanzee GAGAGCCUGCUAAGUGAUU AAUCACUUAGCAGGCUCUC[279-297](19/19) [283-295](13/13) [164-176](13/13) [399-417](19/19)[263-279] (16/17) 559 human GCAUAGAUCCCAUUUUUGU ACAAAAAUGGGAUCUAUGC[998-1016](19/19) — — — — 560 human AGUGUAGAUUUUCUGCAUAUAUGCAGAAAAUCUACACU [984-1002](19/19) — — — — 561 humanUGGAGAAGUGAUUCAAAAU AUUUUGAAUCACUUCUCCA [965-983](19/19) — — — — 562human ACUGCAGCUAACAGGCUGA UCAGCCUGUUAGCUGCAGU [934-952](19/19) — — — —563 human CUGUGUUUCACAUUCAUAG CUAUGAAUGUGAAACACAG [913-931](19/19) — — —— 564 human UUCUGUGUUUCACAUUCAU AUGAAUGUGAAACACAGAA [911-929](19/19) — —— — 565 human CCCAAAUGUAGUCUCUUUU AAAAGAGACUACAUUUGGG [890-908](19/19) —— — — 566 human CCCCAAAUGUAGUCUCUUU AAAGAGACUACAUUUGGGG [889-907](19/19)— — — — 567 human AACCCCAAAUGUAGUCUCU AGAGACUACAUUUGGGGUU[887-905](19/19) — — — — 568 human CUGCCCUAAAUAAGAAACCGGUUUCUUAUUUAGGGCAG [872-890](19/19) — — — — 569 humanACCUGCCCUAAAUAAGAAA UUUCUUAUUUAGGGCAGGU [870-888](19/19) — — — — 570human CCCACCUGCCCUAAAUAAG CUUAUUUAGGGCAGGUGGG [867-885](19/19) — — — —571 human CUCACUGAUUGGAACAACA UGUUGUUCCAAUCAGUGAG [749-767](19/19)[751-761](11/11) [632-642](11/11) — [736-749] (14/14) 572 humanCUCAGGAUUUCGACUUGUU AACAAGUCGAAAUCCUGAG [716-734](19/19)[714-731](18/18) [599-613](15/15) — — 573 human, mouseGCUCAGGAUUUCGACUUGU ACAAGUCGAAAUCCUGAGC [715-733](19/19)[713-731](19/19) [594-612](18/19) — — 574 human, mouseAGCUCAGGAUUUCGACUUG CAAGUCGAAAUCCUGAGCU [714-732](19/19)[712-730](19/19) [593-611](18/19) — — 575 human AGGAGAACUCUGAUCCUCAUGAGGAUCAGAGUUCUCCU [696-714](19/19) — [579-593](15/15) — — 576human, chimpanzee GGCAGUUUGAGCAGCAAGA UCUUGCUGCUCAAACUGCC[216-234](19/19) [214-232](18/19) [95-113](18/19) [336-354](19/19)[198-216] (18/19) 577 human, chimpanzee UGAGCUUACACUUGUGUUUAAACACAAGUGUAAGCUCA [605-623](19/19) — — [725-743](19/19) [588-605](17/18) 578 human, chimpanzee UUUGAGCUUACACUUGUGU ACACAAGUGUAAGCUCAAA[603-621](19/19) — — [723-741](19/19) — 579 human GUGUGUGAUUCUAGCGUCGCGACGCUAGAAUCACACAC [576-594](19/19) — — [696-714](18/19) [558-571](14/14) 580 human UUGUGUGUGAUUCUAGCGU ACGCUAGAAUCACACACAA[574-592](19/19) — [453-468](15/16) [694-709](16/16) [556-571] (16/16)581 human, rat, UGGAUAGGAUUGUGUGUGA UCACACACAAUCCUAUCCA [565-583](19/19)— [444-462](19/19) [685-703](19/19) [547-565] chimpanzee (18/19) 582human, rat, AAAAGCUGGAUAGGAUUGU ACAAUCCUAUCCAGCUUUU [559-577](19/19)[557-572](16/16) [438-456](19/19) [679-697](19/19) [541-559] chimpanzee(18/19) 583 human, mouse, GUAUGUAAAAAGCUGGAUA UAUCCAGCUUUUUACAUAC[552-570](19/19) [550-568](19/19) [434-449](16/16) [672-690](19/19)[534-552] chimpanzee, dog (19/19) 584 human, mouse, AUGUAUGUAAAAAGCUGGAUCCAGCUUUUUACAUACAU [550-568](19/19) [548-566](19/19) [429-447](18/19)[670-688](19/19) [534-550] chimpanzee (17/17) 585 human, mouse,CUGACCCAGAGAAUUGCUC GAGCAAUUCUCUGGGUCAG [453-471](19/19)[451-469](19/19) [332-348](17/17) [573-591](19/19) [435-453](18/19)chimpanzee 586 human, chimpanzee AUGGUUGCAACUGGCAGUU AACUGCCAGUUGCAACCAU[204-222](19/19) [202-220](18/19) [83-101](18/19) [324-342](19/19)[186-204] (18/19) 587 human UGGAAGGCUGUUAAAUUAA UUAAUUUAACAGCCUUCCA[2511-2529](19/19) — — — — 588 human GCUUAUGGAAGGCUGUUAAUUAACAGCCUUCCAUAAGC [2506-2524](19/19) — — — — 589 human, chimpanzee,CUGUCUGUCCAAAUCAAAG CUUUGAUUUGGACAGACAG [392-410](19/19)[390-408](18/19) [271-289](18/19) [512-530](19/19) [374-392] dog (19/19)590 human AUGUAGAACUGUUGUCCUU AAGGACAACAGUUCUACAU [2430-2448](19/19) — —— — 591 human GCAUCGAUGUAGAACUGUU AACAGUUCUACAUCGAUGC [2424-2442](19/19)— — — — 592 human AAUGUUGUUUUGCAUGUCU AGACAUGCAAAACAACAUU[2327-2345](19/19) — — — — 593 human UGGGCCAAGAUAAAUCAAUAUUGAUUUAUCUUGGCCCA [2311-2329](19/19) — — — — 594 humanGAAUUGGGCCAAGAUAAAU AUUUAUCUUGGCCCAAUUC [2307-2325](19/19) — — — — 595human AGAAUUGGGCCAAGAUAAA UUUAUCUUGGCCCAAUUCU [2306-2324](19/19) — — — —596 human CUCUUCAAAUGCUUGGAAA UUUCCAAGCAUUUGAAGAG [2264-2282](19/19) — —— — 597 human ACUCUUCAAAUGCUUGGAA UUCCAAGCAUUUGAAGAGU [2263-2281](19/19)— — — — 598 human GUCACUGUUCAAAUUAGCC GGCUAAUUUGAACAGUGAC[2226-2244](19/19) — — — — 599 human GUGACCUAAAAUGUCACUGCAGUGACAUUUUAGGUCAC [2214-2232](19/19) — — — — 600 humanCUGAUACUAUAAGUGACCU AGGUCACUUAUAGUAUCAG [2202-2220](19/19) — — — — 601human ACUGAUACUAUAAGUGACC GGUCACUUAUAGUAUCAGU [2201-2219](19/19) — — — —602 human UACUGAUACUAUAAGUGAC GUCACUUAUAGUAUCAGUA [2200-2218](19/19) — —— — 603 human GAUCCUGUUACUGAUACUA UAGUAUCAGUAACAGGAUC [2192-2210](19/19)— — — — 604 human GGUGUGAUCCUGUUACUGA UCAGUAACAGGAUCACACC[2187-2205](19/19) — — — — 605 human CUGAAAAACAGGUGUGAUCGAUCACACCUGUUUUUCAG [2177-2195](19/19) — — — — 606 humanUGGUGUUACUGAAAAACAG CUGUUUUUCAGUAACACCA [2169-2187](19/19) — — — — 607human CUGGUGUUACUGAAAAACA UGUUUUUCAGUAACACCAG [2168-2186](19/19) — — — —608 human UCCUGGUGUUACUGAAAAA UUUUUCAGUAACACCAGGA [2166-2184](19/19) — —— — 609 human ACAGAUGUAUUCAUCCUGG CCAGGAUGAAUACAUCUGU [2153-2171](19/19)— — — — 610 human GACAGAUGUAUUCAUCCUG CAGGAUGAAUACAUCUGUC[2152-2170](19/19) — — — — 611 human GCGUAGGGACAGAUGUAUUAAUACAUCUGUCCCUACGC [2145-2163](19/19) — — — — 612 humanUCCUGCCCUAGCUAUUAGC GCUAAUAGCUAGGGCAGGA [2103-2121](19/19) — — — — 613human GUGGAUAAGGAGCUUAUUC GAAUAAGCUCCUUAUCCAC [2079-2097](19/19) — — — —614 human UGCUGUGGGUCGUGGAUAA UUAUCCACGACCCACAGCA [2068-2086](19/19) — —— — 615 human, chimpanzee GAAUCAACUUGCCAGAAUU AAUUCUGGCAAGUUGAUUC[354-372](19/19) — — [474-492](19/19) [340-351] (12/12) 616 humanCCAACUAAGUAGAUCAUUA UAAUGAUCUACUUAGUUGG [1971-1989](19/19) — — — — 617human AAAGGCCUUAUUUUUUGUC GACAAAAAAUAAGGCCUUU [1947-1965](19/19) — — — —618 human GGAAUAAUAAAGGCCUUAU AUAAGGCCUUUAUUAUUCC [1939-1957](19/19) — —— — 619 human GGGAAUAAUAAAGGCCUUA UAAGGCCUUUAUUAUUCCC [1938-1956](19/19)— — — — 620 human AACCAUGCAUGCACCCAGA UCUGGGUGCAUGCAUGGUU[1891-1909](19/19) — — — — 621 human CAGAUAACCAUGCAUGCACGUGCAUGCAUGGUUAUCUG [1886-1904](19/19) — — — — 622 humanCUCACCGUCCAGAUAACCA UGGUUAUCUGGACGGUGAG [1877-1895](19/19) — — — — 623human AUGAAACUCACCGUCCAGA UCUGGACGGUGAGUUUCAU [1871-1889](19/19) — — — —624 human AGAGAAUGAAACUCACCGU ACGGUGAGUUUCAUUCUCU [1866-1884](19/19) — —— — 625 human GUACAGCCUAGAGAAUGAA UUCAUUCUCUAGGCUGUAC [1857-1875](19/19)— — — — 626 human AUGGUUUAUAGUACAGCCU AGGCUGUACUAUAAACCAU[1847-1865](19/19) — — — — 627 human AUGGAGAUAUGGUUUAUAGCUAUAAACCAUAUCUCCAU [1839-1857](19/19) — — — — 628 humanUGGCUAUGGAGAUAUGGUU AACCAUAUCUCCAUAGCCA [1834-1852](19/19) — — — — 629human UGCAUUGGCUAUGGAGAUA UAUCUCCAUAGCCAAUGCA [1829-1847](19/19) — — — —630 human GUAUACUACCACUUUGAAU AUUCAAAGUGGUAGUAUAC [1783-1801](19/19) — —— — 631 human AGCUGUAUACUACCACUUU AAAGUGGUAGUAUACAGCU [1779-1797](19/19)— — — — 632 human GGUAGUAGCUGUAUACUAC GUAGUAUACAGCUACUACC[1773-1791](19/19) — — — — 633 human CAAGGGUAGUAGCUGUAUAUAUACAGCUACUACCCUUG [1769-1787](19/19) — — — — 634 humanUCCUUAACCCCAAUUGUCA UGACAAUUGGGGUUAAGGA [1752-1770](19/19) — — — — 635human AAGUAUCUCUCCUUAACCC GGGUUAAGGAGAGAUACUU [1743-1761](19/19) — — — —636 human UCUGGGAAUUGAAGUAUCU AGAUACUUCAAUUCCCAGA [1732-1750](19/19) — —— — 637 human UUUCUGGGAAUUGAAGUAU AUACUUCAAUUCCCAGAAA [1730-1748](19/19)— — — — 638 human GGCAGUGUUAUCUCAUCUC GAGAUGAGAUAACACUGCC[1705-1723](19/19) — — — — 639 human AUGGCAGUGUUAUCUCAUCGAUGAGAUAACACUGCCAU [1703-1721](19/19) — — — — 640 humanCACCAUGGCAGUGUUAUCU AGAUAACACUGCCAUGGUG [1699-1717](19/19) — — — — 641human UGCUUAAUCUCAGAUGAAC GUUCAUCUGAGAUUAAGCA [1675-1693](19/19) — — — —642 human CAUGUGCUUAAUCUCAGAU AUCUGAGAUUAAGCACAUG [1671-1689](19/19) — —— — 643 human UUACAUGUGCUUAAUCUCA UGAGAUUAAGCACAUGUAA [1668-1686](19/19)— — — — 644 human CAGUAUUACAUGUGCUUAA UUAAGCACAUGUAAUACUG[1663-1681](19/19) — — — — 645 human UCACUUAGUAAUCUAUCCUAGGAUAGAUUACUAAGUGA [1639-1657](19/19) — — — — 646 humanCUGUGAGGAUAGGAAAUUA UAAUUUCCUAUCCUCACAG [1594-1612](19/19) — — — — 647human AGUGUUGAAUACUGUCUUU AAAGACAGUAUUCAACACU [1496-1514](19/19) — — — —648 human UAAGUGUUGAAUACUGUCU AGACAGUAUUCAACACUUA [1494-1512](19/19) — —— — 649 human CUGUUCUUAAGUGUUGAAU AUUCAACACUUAAGAACAG [1487-1505](19/19)— — — — 650 human CCUGUUCUUAAGUGUUGAA UUCAACACUUAAGAACAGG[1486-1504](19/19) — — — — 651 human GAUGCCUUUAUAAGCUCAGCUGAGCUUAUAAAGGCAUC [1462-1480](19/19) — — — — 652 humanUAGAUGCCUUUAUAAGCUC GAGCUUAUAAAGGCAUCUA [1460-1478](19/19) — — — — 653human GGUGUUGUUUUAGAUGCCU AGGCAUCUAAAACAACACC [1450-1468](19/19) — — — —654 human UAAAGAGGCAACAAAAGCU AGCUUUUGUUGCCUCUUUA [1408-1426](19/19) — —— — 655 human GUUUUAAAGAGGCAACAAA UUUGUUGCCUCUUUAAAAC [1404-1422](19/19)— — — — 656 human AUGUUUUAAAGAGGCAACA UGUUGCCUCUUUAAAACAU[1402-1420](19/19) — — — — 657 human GCUUCACUGUUUCUUGGUGCACCAAGAAACAGUGAAGC [1369-1387](19/19) — — — [1333-1346] (14/14) 658human GAGAAAUAUUACGGCAAUA UAUUGCCGUAAUAUUUCUC [1342-1360](19/19) — — — —659 human ACUCAAAUUUGAAGGGUUU AAACCCUUCAAAUUUGAGU [1258-1276](19/19) — —— — 660 human, chimpanzee AAGUGAUUUUGACUACUGG CCAGUAGUCAAAAUCACUU[290-308](19/19) — — [410-428](19/19) [275-290] (16/16) 661human, chimpanzee UAAGUGAUUUUGACUACUG CAGUAGUCAAAAUCACUUA[289-307](19/19) — — [409-427](19/19) [275-289] (15/15) 662 humanCACAGAAUCAUACUAAAUG CAUUUAGUAUGAUUCUGUG [1192-1210](19/19) — — — — 663human, chimpanzee GCUAAGUGAUUUUGACUAC GUAGUCAAAAUCACUUAGC[287-305](19/19) [285-303](18/19) [166-184](18/19) [407-425](19/19)[269-287] (18/19) 664 human CAGGUAGGCUUGGUAAUAG CUAUUACCAAGCCUACCUG[1096-1114](19/19) — — — — 665 human UGGGAUGGAAUAGGUAAGCGCUUACCUAUUCCAUCCCA [1028-1046](19/19) — — — — 666 humanAUGGGAUGGAAUAGGUAAG CUUACCUAUUCCAUCCCAU [1027-1045](19/19) — — — — 667human UAGUCUCUUUUCUUUCUGU ACAGAAAGAAAAGAGACUA [898-916](19/19) — — — —668 human AAGAAACCCCAAAUGUAGU ACUACAUUUGGGGUUUCUU [883-901](19/19) — — —[866-878] (13/13) 669 human CCCUAAAUAAGAAACCCCA UGGGGUUUCUUAUUUAGGG[875-893](19/19) — — — — 670 human GAACAACAGUGAUUGAAGGCCUUCAAUCACUGUUGUUC [760-778](19/19) — — — — 671 humanCUGAUUGGAACAACAGUGA UCACUGUUGUUCCAAUCAG [753-771](19/19)[751-767](16/17) [632-648](16/17) — [736-751] (16/16) 672 humanGAUCCUCAGCUCAGGAUUU AAAUCCUGAGCUGAGGAUC [707-725](19/19)[709-723](15/15) [586-604](18/19) — — 673 human UUCAGGAGAACUCUGAUCCGGAUCAGAGUUCUCCUGAA [693-711](19/19) — [579-590](12/12) — — 674 humanUAGUAGAGGUCGCUUCUCC GGAGAAGCGACCUCUACUA [668-686](19/19)[670-684](15/15) [552-564](13/13) — [657-667] (11/11) 675human, chimpanzee GCUUCAGGGACUUUUUCUU AAGAAAAAGUCCCUGAAGC[649-667](19/19) — — [769-787](19/19) — 676 human, chimpanzee,AAGCAGGAGAACUGCUCAU AUGAGCAGUUCUCCUGCUU [624-642](19/19) — —[744-762](19/19) [606-624] dog (19/19) 677 human, chimpanzeeGAGCUUACACUUGUGUUUA UAAACACAAGUGUAAGCUC [606-624](19/19) — —[726-744](19/19) [588-606] (18/19) 678 human GUGUGAUUCUAGCGUCGUAUACGACGCUAGAAUCACAC [578-596](19/19) — — [698-715](17/18) [560-571](12/12) 679 human, chimpanzee AGGAUUGUGUGUGAUUCUA UAGAAUCACACACAAUCCU[570-588](19/19) — [449-463](15/15) [690-708](19/19) [554-570] (17/17)680 human, chimpanzee GGUCCUUGUCCCUGAGAAA UUUCUCAGGGACAAGGACC[434-452](19/19) [439-450](12/12) — [554-572](19/19) [417-434] (18/18)681 human ACAAAAUGGUGAUGGCUUA UAAGCCAUCACCAUUUUGU [2492-2510](19/19) — —— — 682 human CUCUCUUGCCUGUUAUGCU AGCAUAACAGGCAAGAGAG [2472-2490](19/19)— — — — 683 human AUCUUUGACUCUCUUGCCU AGGCAAGAGAGUCAAAGAU[2464-2482](19/19) — — — — 684 human UUCCACUAACAGUUAUCUUAAGAUAACUGUUAGUGGAA [2450-2468](19/19) — — — — 685 humanUCGAUGUAGAACUGUUGUC GACAACAGUUCUACAUCGA [2427-2445](19/19) — — — — 686human UCUUGGGCAUCGAUGUAGA UCUACAUCGAUGCCCAAGA [2418-2436](19/19) — — — —687 human AAGGCUUCUUGGGCAUCGA UCGAUGCCCAAGAAGCCUU [2412-2430](19/19) — —— — 688 human GUCUAUUGUUAAGCUCCAA UUGGAGCUUAACAAUAGAC [2342-2360](19/19)— — — — 689 human UGUCUAUUGUUAAGCUCCA UGGAGCUUAACAAUAGACA[2341-2359](19/19) — — — — 690 human UGCAUGUCUAUUGUUAAGCGCUUAACAAUAGACAUGCA [2337-2355](19/19) — — — — 691 humanUGUUUUGCAUGUCUAUUGU ACAAUAGACAUGCAAAACA [2332-2350](19/19) — — — — 692human UUGUUUUGCAUGUCUAUUG CAAUAGACAUGCAAAACAA [2331-2349](19/19) — — — —693 human AGUGACCUAAAAUGUCACU AGUGACAUUUUAGGUCACU [2213-2231](19/19) — —— — 694 human GUUACUGAUACUAUAAGUG CACUUAUAGUAUCAGUAAC [2198-2216](19/19)— — — — 695 human AAACAGGUGUGAUCCUGUU AACAGGAUCACACCUGUUU[2182-2200](19/19) — — — — 696 human UCAUCCUGGUGUUACUGAAUUCAGUAACACCAGGAUGA [2163-2181](19/19) — — — — 697 humanGAUGUAUUCAUCCUGGUGU ACACCAGGAUGAAUACAUC [2156-2174](19/19) — — — — 698human CUAGCUAUUAGCUCCACUU AAGUGGAGCUAAUAGCUAG [2110-2128](19/19) — — — —699 human UAAGGAGCUUAUUCAGGUU AACCUGAAUAAGCUCCUUA [2084-2102](19/19) — —— — 700 human GUCGUGGAUAAGGAGCUUA UAAGCUCCUUAUCCACGAC [2076-2094](19/19)— — — — 701 human UGUGGGUCGUGGAUAAGGA UCCUUAUCCACGACCCACA[2071-2089](19/19) — — — — 702 human UUCAUAUCCUUGCUGUGGGCCCACAGCAAGGAUAUGAA [2058-2076](19/19) — — — — 703 humanUAAUGAGAGAAUUUAGCCU AGGCUAAAUUCUCUCAUUA [2009-2027](19/19) — — — — 704human GUUAAUGAGAGAAUUUAGC GCUAAAUUCUCUCAUUAAC [2007-2025](19/19) — — — —705 human CUUAUUCCAACUAAGUAGA UCUACUUAGUUGGAAUAAG [1965-1983](19/19) — —— — 706 human UUGUCUUAUUCCAACUAAG CUUAGUUGGAAUAAGACAA [1961-1979](19/19)— — — — 707 human GUCACUAGGGAAUAAUAAA UUUAUUAUUCCCUAGUGAC[1931-1949](19/19) — — — — 708 human CUGUCACUAGGGAAUAAUAUAUUAUUCCCUAGUGACAG [1929-1947](19/19) — — — — 709 humanCGUCCAGAUAACCAUGCAU AUGCAUGGUUAUCUGGACG [1882-1900](19/19) — — — — 710human UAGAGAAUGAAACUCACCG CGGUGAGUUUCAUUCUCUA [1865-1883](19/19) — — — —711 human CUACCACUUUGAAUUAUUG CAAUAAUUCAAAGUGGUAG [1788-1806](19/19) — —— — 712 human UGUCAAGGGUAGUAGCUGU ACAGCUACUACCCUUGACA [1766-1784](19/19)— — — — 713 human CUCUCCUUAACCCCAAUUG CAAUUGGGGUUAAGGAGAG[1749-1767](19/19) — — — — 714 human GAUGAACCAUUUCACCAUGCAUGGUGAAAUGGUUCAUC [1687-1705](19/19) — — — — 715 humanUUUCAGUAUUACAUGUGCU AGCACAUGUAAUACUGAAA [1660-1678](19/19) — — — — 716human, chimpanzee UUCCUGAACCCAACCUCAA UUGAGGUUGGGUUCAGGAA[319-337](19/19) [319-335](16/17) [206-216](11/11) [439-457](19/19)[301-319] (18/19) 717 human GUAAUCUAUCCUCUUUUCA UGAAAAGAGGAUAGAUUAC[1646-1664](19/19) — — — — 718 human, chimpanzee UUGUUCCUGAACCCAACCUAGGUUGGGUUCAGGAACAA [316-334](19/19) — — [436-454](19/19) [298-316](18/19) 719 human GGAAAUUAGUUCUGAGAUC GAUCUCAGAACUAAUUUCC[1605-1623](19/19) — — — — 720 human AGGAUAGGAAAUUAGUUCUAGAACUAAUUUCCUAUCCU [1599-1617](19/19) — — — — 721 humanUGUGAGGAUAGGAAAUUAG CUAAUUUCCUAUCCUCACA [1595-1613](19/19) — — — — 722human CCUGUGAGGAUAGGAAAUU AAUUUCCUAUCCUCACAGG [1593-1611](19/19) — — — —723 human CUGAUAUUUUUGUGUGUAG CUACACACAAAAAUAUCAG [1541-1559](19/19) — —— — 724 human GUUCUUAAGUGUUGAAUAC GUAUUCAACACUUAAGAAC [1489-1507](19/19)— — — — 725 human, chimpanzee ACUGGGAUUAUGUUGUUCC GGAACAACAUAAUCCCAGU[304-322](19/19) [302-317](16/16) [183-195](13/13) [424-442](19/19) —726 human GGUGACUUCCUCACUCUAA UUAGAGUGAGGAAGUCACC [1384-1402](19/19) — —— — 727 human UUGGUGACUUCCUCACUCU AGAGUGAGGAAGUCACCAA [1382-1400](19/19)— — — — 728 human CACUGUUUCUUGGUGACUU AAGUCACCAAGAAACAGUG[1373-1391](19/19) — — — [1336-1346] (11/11) 729 humanCUUCACUGUUUCUUGGUGA UCACCAAGAAACAGUGAAG [1370-1388](19/19) — — —[1333-1346] (14/14) 730 human UAUUACGGCAAUAAUGGAA UUCCAUUAUUGCCGUAAUA[1348-1366](19/19) — — — — 731 human UAGGUAAGAGUAAAUGAGAUCUCAUUUACUCUUACCUA [1327-1345](19/19) — — — — 732 humanCCCUAGGUAAGAGUAAAUG CAUUUACUCUUACCUAGGG [1324-1342](19/19) — — — — 733human UUAGACAGGAAGGUAGGAU AUCCUACCUUCCUGUCUAA [1277-1295](19/19) — — — —734 human, chimpanzee CUAAGUGAUUUUGACUACU AGUAGUCAAAAUCACUUAG[288-306](19/19) [286-304](18/19) [167-185](18/19) [408-426](19/19)[270-288] (18/19) 735 human AGAAGCCCAUUUGAGUUUU AAAACUCAAAUGGGCUUCU[1053-1071](19/19) — — — — 736 human GUAGAAGCCCAUUUGAGUUAACUCAAAUGGGCUUCUAC [1051-1069](19/19) — — — — 737 humanAAGUAGAAGCCCAUUUGAG CUCAAAUGGGCUUCUACUU [1049-1067](19/19) — — — — 738human CAAAAGUAGAAGCCCAUUU AAAUGGGCUUCUACUUUUG [1046-1064](19/19) — — — —739 human, chimpanzee AGCCUGCUAAGUGAUUUUG CAAAAUCACUUAGCAGGCU[282-300](19/19) [283-296](14/14) [164-177](14/14) [402-420](19/19) —740 human GGUAAGCAAAAGUAGAAGC GCUUCUACUUUUGCUUACC [1040-1058](19/19) — —— — 741 human AGGUAAGCAAAAGUAGAAG CUUCUACUUUUGCUUACCU [1039-1057](19/19)— — — — 742 human UGGAAUAGGUAAGCAAAAG CUUUUGCUUACCUAUUCCA[1033-1051](19/19) — — — — 743 human AUGGAAUAGGUAAGCAAAAUUUUGCUUACCUAUUCCAU [1032-1050](19/19) — — — — 744 humanUUGAAUGGGAUGGAAUAGG CCUAUUCCAUCCCAUUCAA [1023-1041](19/19) — — — — 745human, chimpanzee AGAGCCUGCUAAGUGAUUU AAAUCACUUAGCAGGCUCU[280-298](19/19) [283-296](14/14) [164-177](14/14) [400-418](19/19)[263-280] (17/18) 746 human UCUGCAUAGAUCCCAUUUU AAAAUGGGAUCUAUGCAGA[995-1013](19/19) — — — — 747 human UUCUGGCCUUUGGAGAAGUACUUCUCCAAAGGCCAGAA [955-973](19/19) — — — — 748 humanUUUCUGUGUUUCACAUUCA UGAAUGUGAAACACAGAAA [910-928](19/19) — — — — 749human GUAGUCUCUUUUCUUUCUG CAGAAAGAAAAGAGACUAC [897-915](19/19) — — — —750 human GAAACCCCAAAUGUAGUCU AGACUACAUUUGGGGUUUC [885-903](19/19) — — —[867-878] (12/12) 751 human GUAGUAAAACUAUUCAGCU AGCUGAAUAGUUUUACUAC[821-839](19/19) — — — — 752 human GGUAGUAAAACUAUUCAGCGCUGAAUAGUUUUACUACC [820-838](19/19) — — — — 753 humanGAUUGGAACAACAGUGAUU AAUCACUGUUGUUCCAAUC [755-773](19/19) — — — [737-751](15/15) 754 human UACUCACUGAUUGGAACAA UUGUUCCAAUCAGUGAGUA[747-765](19/19) [745-761](16/17) [626-642](16/17) — [736-747] (12/12)755 human, rat GAUUUCGACUUGUUAAGAA UUCUUAACAAGUCGAAAUC [721-739](19/19)[719-737](18/19) [600-618](19/19) — — 756 human AGGAUUUCGACUUGUUAAGCUUAACAAGUCGAAAUCCU [719-737](19/19) [717-731](15/15) [599-616](18/18) —— 757 human UCAGGAGAACUCUGAUCCU AGGAUCAGAGUUCUCCUGA [694-712](19/19) —[579-591](13/13) — — 758 human, chimpanzee GGACUAGCUUCAGGGACUUAAGUCCCUGAAGCUAGUCC [643-661](19/19) — — [763-781](19/19) [625-640](16/16) 759 human, chimpanzee, CUCAUGGACUAGCUUCAGG CCUGAAGCUAGUCCAUGAG[638-656](19/19) — — [758-776](19/19) [620-638] dog (19/19) 760human, chimpanzee, GAGAACUGCUCAUGGACUA UAGUCCAUGAGCAGUUCUC[630-648](19/19) — — [750-768](19/19) [612-630] dog (19/19) 761human, chimpanzee, GGAGAACUGCUCAUGGACU AGUCCAUGAGCAGUUCUCC[629-647](19/19) — — [749-767](19/19) [611-629] dog (19/19) 762human, chimpanzee, CAGGAGAACUGCUCAUGGA UCCAUGAGCAGUUCUCCUG[627-645](19/19) — — [747-765](19/19) [609-627] dog (19/19) 763human, chimpanzee GCUUACACUUGUGUUUAAG CUUAAACACAAGUGUAAGC[608-626](19/19) [613-624](12/12) — [728-746](19/19) [594-608] (15/15)764 human, chimpanzee GGAUUGUGUGUGAUUCUAG CUAGAAUCACACACAAUCC[571-589](19/19) — [450-468](18/19) [691-709](19/19) [554-571] (18/18)765 human, chimpanzee GGAUAGGAUUGUGUGUGAU AUCACACACAAUCCUAUCC[566-584](19/19) — [445-463](19/19) [686-704](19/19) [548-566] (18/19)766 human, chimpanzee GGUUGCAACUGGCAGUUUG CAAACUGCCAGUUGCAACC[206-224](19/19) — — [326-344](19/19) [192-206] (15/15) 767human, chimpanzee UAAACUUGGUUGCUCAAAG CUUUGAGCAACCAAGUUUA[416-434](19/19) [415-428](14/14) — [536-554](19/19) [402-415] (14/14)768 human GGCUUAUGGAAGGCUGUUA UAACAGCCUUCCAUAAGCC [2505-2523](19/19) — —— — 769 human AAAUGGUGAUGGCUUAUGG CCAUAAGCCAUCACCAUUU [2495-2513](19/19)— — — — 770 human UCUCUUGCCUGUUAUGCUU AAGCAUAACAGGCAAGAGA[2473-2491](19/19) — — — — 771 human UUCUUGGGCAUCGAUGUAGCUACAUCGAUGCCCAAGAA [2417-2435](19/19) — — — — 772 humanAAGCUCCAAAGGUUCACUG CAGUGAACCUUUGGAGCUU [2352-2370](19/19) — — — — 773human UCUAUUGUUAAGCUCCAAA UUUGGAGCUUAACAAUAGA [2343-2361](19/19) — — — —774 human GGGCCAAGAUAAAUCAAUG CAUUGAUUUAUCUUGGCCC [2312-2330](19/19) — —— — 775 human UAGAAUUGGGCCAAGAUAA UUAUCUUGGCCCAAUUCUA [2305-2323](19/19)— — — — 776 human GGAAAGAUACUACAAAGCC GGCUUUGUAGUAUCUUUCC[2278-2296](19/19) [1571-1581] — — — (11/11) 777 humanUCAAAUGCUUGGAAAGAUA UAUCUUUCCAAGCAUUUGA [2268-2286](19/19) — — — — 778human GUUACUGAAAAACAGGUGU ACACCUGUUUUUCAGUAAC [2173-2191](19/19) — — — —779 human AUGUAUUCAUCCUGGUGUU AACACCAGGAUGAAUACAU [2157-2175](19/19) — —— — 780 human CAGAUGUAUUCAUCCUGGU ACCAGGAUGAAUACAUCUG [2154-2172](19/19)— — — — 781 human UGCCCUAGCUAUUAGCUCC GGAGCUAAUAGCUAGGGCA[2106-2124](19/19) — — — — 782 human GAGCUUAUUCAGGUUUCCUAGGAAACCUGAAUAAGCUC [2088-2106](19/19) — — — — 783 humanGGAGCUUAUUCAGGUUUCC GGAAACCUGAAUAAGCUCC [2087-2105](19/19) — — — — 784human UCAUAUCCUUGCUGUGGGU ACCCACAGCAAGGAUAUGA [2059-2077](19/19) — — — —785 human GUUCAAUUCAGCAAGGCUU AAGCCUUGCUGAAUUGAAC [2040-2058](19/19)[2152-2167] — — — (15/16) 786 human CAACAAUGUUCAAUUCAGCGCUGAAUUGAACAUUGUUG [2033-2051](19/19) — — — — 787 humanGUCUUAUUCCAACUAAGUA UACUUAGUUGGAAUAAGAC [1963-1981](19/19) — — — — 788human CUAGGGAAUAAUAAAGGCC GGCCUUUAUUAUUCCCUAG [1935-1953](19/19) — — — —789 human UAACCAUGCAUGCACCCAG CUGGGUGCAUGCAUGGUUA [1890-1908](19/19) — —— — 790 human AGAUAACCAUGCAUGCACC GGUGCAUGCAUGGUUAUCU [1887-1905](19/19)— — — — 791 human AGCCUAGAGAAUGAAACUC GAGUUUCAUUCUCUAGGCU[1861-1879](19/19) — — — — 792 human UUAUAGUACAGCCUAGAGAUCUCUAGGCUGUACUAUAA [1852-1870](19/19) — — — — 793 humanGCUAUGGAGAUAUGGUUUA UAAACCAUAUCUCCAUAGC [1836-1854](19/19) — — — — 794human UCUCUCCUUAACCCCAAUU AAUUGGGGUUAAGGAGAGA [1748-1766](19/19) — — — —795 human AUCUCUCCUUAACCCCAAU AUUGGGGUUAAGGAGAGAU [1747-1765](19/19) — —— — 796 human CUGGGCUUUUCUGGGAAUU AAUUCCCAGAAAAGCCCAG [1723-1741](19/19)— — — — 797 human AACCAUUUCACCAUGGCAG CUGCCAUGGUGAAAUGGUU[1691-1709](19/19) — — — — 798 human UUCAGUAUUACAUGUGCUUAAGCACAUGUAAUACUGAA [1661-1679](19/19) — — — — 799 humanUCCUCUUUUCAGUAUUACA UGUAAUACUGAAAAGAGGA [1654-1672](19/19) — — — — 800human AUCCUCUUUUCAGUAUUAC GUAAUACUGAAAAGAGGAU [1653-1671](19/19) — — — —801 human CUAUCCUCUUUUCAGUAUU AAUACUGAAAAGAGGAUAG [1651-1669](19/19) — —— — 802 human AAUCUAUCCUCUUUUCAGU ACUGAAAAGAGGAUAGAUU [1648-1666](19/19)— — — — 803 human UUCACUUAGUAAUCUAUCC GGAUAGAUUACUAAGUGAA[1638-1656](19/19) — — — — 804 human UAGGAAAUUAGUUCUGAGAUCUCAGAACUAAUUUCCUA [1603-1621](19/19) — — — — 805 humanGGAUAGGAAAUUAGUUCUG CAGAACUAAUUUCCUAUCC [1600-1618](19/19) — — — — 806human GAGGAUAGGAAAUUAGUUC GAACUAAUUUCCUAUCCUC [1598-1616](19/19) — — — —807 human GUGAGGAUAGGAAAUUAGU ACUAAUUUCCUAUCCUCAC [1596-1614](19/19) — —— — 808 human GUAGGCUUGGUAAUAGACU AGUCUAUUACCAAGCCUAC [1099-1117](19/19)— — — — 809 human, chimpanzee CCUGCUAAGUGAUUUUGAC GUCAAAAUCACUUAGCAGG[284-302](19/19) [283-296](14/14) [164-177](14/14) [404-422](19/19) —810 human UGCAUAGAUCCCAUUUUUG CAAAAAUGGGAUCUAUGCA [997-1015](19/19) — —— — 811 human UUCUUUCUGUGUUUCACAU AUGUGAAACACAGAAAGAA [907-925](19/19) —— — — 812 human CAAAUGUAGUCUCUUUUCU AGAAAAGAGACUACAUUUG [892-910](19/19)— — — — 813 human AAACCCCAAAUGUAGUCUC GAGACUACAUUUGGGGUUU[886-904](19/19) — — — [868-878] (11/11) 814 human UGCCCUAAAUAAGAAACCCGGGUUUCUUAUUUAGGGCA [873-891](19/19) — — — — 815 humanAAACUAUUCAGCUAGUCAG CUGACUAGCUGAAUAGUUU [827-845](19/19) — — — — 816human GUGAUUGAAGGGUCCUAAA UUUAGGACCCUUCAAUCAC [768-786](19/19) — — — —817 human UCAGGAUUUCGACUUGUUA UAACAAGUCGAAAUCCUGA [717-735](19/19)[715-731](17/17) [599-614](16/16) — — 818 human UAGAGGUCGCUUCUCCUCUAGAGGAGAAGCGACCUCUA [671-689](19/19) [670-686](17/17) [552-564](13/13) —[657-667] (11/11) 819 human, chimpanzee, AACUGCUCAUGGACUAGCUAGCUAGUCCAUGAGCAGUU [633-651](19/19) — — [753-771](19/19) [615-633] dog(19/19) 820 human GGUGAUGGCUUAUGGAAGG CCUUCCAUAAGCCAUCACC[2499-2517](19/19) — — — — 821 human AGGCUUCUUGGGCAUCGAUAUCGAUGCCCAAGAAGCCU [2413-2431](19/19) — — — — 822 humanUAAGCUCCAAAGGUUCACU AGUGAACCUUUGGAGCUUA [2351-2369](19/19) — — — — 823human GGCCAAGAUAAAUCAAUGU ACAUUGAUUUAUCUUGGCC [2313-2331](19/19) — — — —824 human UUCAAAUGCUUGGAAAGAU AUCUUUCCAAGCAUUUGAA [2267-2285](19/19) — —— — 825 human GGGACAGAUGUAUUCAUCC GGAUGAAUACAUCUGUCCC [2150-2168](19/19)— — — — 826 human AGCUUAUUCAGGUUUCCUG CAGGAAACCUGAAUAAGCU[2089-2107](19/19) — — — — 827 human AUUCCAACUAAGUAGAUCAUGAUCUACUUAGUUGGAAU [1968-1986](19/19) — — — — 828 humanAGUACAGCCUAGAGAAUGA UCAUUCUCUAGGCUGUACU [1856-1874](19/19) — — — — 829human AUAGUACAGCCUAGAGAAU AUUCUCUAGGCUGUACUAU [1854-1872](19/19) — — — —830 human GGUUUAUAGUACAGCCUAG CUAGGCUGUACUAUAAACC [1849-1867](19/19) — —— — 831 human GAUAUGGUUUAUAGUACAG CUGUACUAUAAACCAUAUC [1844-1862](19/19)— — — — 832 human GGAGAUAUGGUUUAUAGUA UACUAUAAACCAUAUCUCC[1841-1859](19/19) — — — — 833 human AACCCCAAUUGUCAAGGGUACCCUUGACAAUUGGGGUU [1757-1775](19/19) — — — — 834 humanUAUCUCUCCUUAACCCCAA UUGGGGUUAAGGAGAGAUA [1746-1764](19/19) — — — — 835human GUAUCUCUCCUUAACCCCA UGGGGUUAAGGAGAGAUAC [1745-1763](19/19) — — — —836 human UCUGGGCUUUUCUGGGAAU AUUCCCAGAAAAGCCCAGA [1722-1740](19/19) — —— — 837 human GAACCAUUUCACCAUGGCA UGCCAUGGUGAAAUGGUUC [1690-1708](19/19)— — — — 838 human UCUAUCCUCUUUUCAGUAU AUACUGAAAAGAGGAUAGA[1650-1668](19/19) — — — — 839 human UGUGUGUAGUUGAUUACUCGAGUAAUCAACUACACACA [1551-1569](19/19) — — — — 840 humanCUUAAGUGUUGAAUACUGU ACAGUAUUCAACACUUAAG [1492-1510](19/19) — — — — 841human UCUUAAGUGUUGAAUACUG CAGUAUUCAACACUUAAGA [1491-1509](19/19) — — — —842 human AAACCAGAUUUGCCUAUUU AAAUAGGCAAAUCUGGUUU [1122-1140](19/19) — —— — 843 human CCUUUGGAGAAGUGAUUCA UGAAUCACUUCUCCAAAGG [961-979](19/19) —— — — 844 human UGGCCUUUGGAGAAGUGAU AUCACUUCUCCAAAGGCCA [958-976](19/19)— — — — 845 human UGUAGUCUCUUUUCUUUCU AGAAAGAAAAGAGACUACA[896-914](19/19) — — — — 846 human CUAAAUAAGAAACCCCAAAUUUGGGGUUUCUUAUUUAG [877-895](19/19) — — — [866-877] (12/12) 847 humanCCUGCCCUAAAUAAGAAAC GUUUCUUAUUUAGGGCAGG [871-889](19/19) — — — — 848human AAACUUUACUCACUGAUUG CAAUCAGUGAGUAAAGUUU [741-759](19/19) — — — —849 human GAAAAAACUUUACUCACUG CAGUGAGUAAAGUUUUUUC [737-755](19/19) — — —— 850 human, rat GGAUUUCGACUUGUUAAGA UCUUAACAAGUCGAAAUCC[720-738](19/19) [718-736](18/19) [599-617](19/19) — — 851 humanCAGGAUUUCGACUUGUUAA UUAACAAGUCGAAAUCCUG [718-736](19/19)[716-731](16/16) [599-615](17/17) — — 852 human, chimpanzeeCUGGCAGUUUGAGCAGCAA UUGCUGCUCAAACUGCCAG [214-232](19/19)[214-227](14/14) [95-108](14/14) [334-352](19/19) [196-211] (16/16) 853human, chimpanzee ACUGGCAGUUUGAGCAGCA UGCUGCUCAAACUGCCAGU[213-231](19/19) [214-227](14/14) [95-108](14/14) [333-351](19/19)[195-211] (17/17) 854 human, chimpanzee UAGGAUUGUGUGUGAUUCUAGAAUCACACACAAUCCUA [569-587](19/19) — [448-463](16/16) [689-707](19/19)[554-569] (16/16) 855 human, rat, CUGGAUAGGAUUGUGUGUGCACACACAAUCCUAUCCAG [564-582](19/19) [562-580](18/19) [443-461](19/19)[684-702](19/19) [546-564] chimpanzee (18/19) 856 human, chimpanzeeCAAAGGUCCUUGUCCCUGA UCAGGGACAAGGACCUUUG [430-448](19/19) — —[550-568](19/19) [412-430] (18/19) 857 human, chimpanzeeGUUGCUCAAAGGUCCUUGU ACAAGGACCUUUGAGCAAC [424-442](19/19)[597-607](11/11) [478-488](11/11) [544-562](19/19) — 858human, chimpanzee AAACUUGGUUGCUCAAAGG CCUUUGAGCAACCAAGUUU[417-435](19/19) [415-433](18/19) — [537-555](19/19) [402-415] (14/14)859 human UGGUGAUGGCUUAUGGAAG CUUCCAUAAGCCAUCACCA [2498-2516](19/19) — —— — 860 human GGAGUUGUCACCACUGACU AGUCAGUGGUGACAACUCC [2389-2407](19/19)— — — — 861 human ACAAUGUUCAAUUCAGCAA UUGCUGAAUUGAACAUUGU[2035-2053](19/19) — — — — 862 human, chimpanzee UGAGGAAUCAACUUGCCAGCUGGCAAGUUGAUUCCUCA [350-368](19/19) — — [470-468](19/19) [340-350](11/11) 863 human UGGAGAUAUGGUUUAUAGU ACUAUAAACCAUAUCUCCA[1840-1858](19/19) — — — — 864 human CUAUGGAGAUAUGGUUUAUAUAAACCAUAUCUCCAUAG [1837-1855](19/19) — — — — 865 humanCCCCAAUUGUCAAGGGUAG CUACCCUUGACAAUUGGGG [1759-1777](19/19) — — — — 866human UAACCCCAAUUGUCAAGGG CCCUUGACAAUUGGGGUUA [1756-1774](19/19) — — — —867 human ACUUAGUAAUCUAUCCUCU AGAGGAUAGAUUACUAAGU [1641-1659](19/19) — —— — 868 human CACUUAGUAAUCUAUCCUC GAGGAUAGAUUACUAAGUG [1640-1658](19/19)— — — — 869 human AGGAAAUUAGUUCUGAGAU AUCUCAGAACUAAUUUCCU[1604-1622](19/19) — — — — 870 human UGGUGACUUCCUCACUCUAUAGAGUGAGGAAGUCACCA [1383-1401](19/19) — — — — 871 humanGUUUCAGGUAGGCUUGGUA UACCAAGCCUACCUGAAAC [1092-1110](19/19) — — — — 872human AUAGGUAAGCAAAAGUAGA UCUACUUUUGCUUACCUAU [1037-1055](19/19) — — — —873 human ACAACAGUGAUUGAAGGGU ACCCUUCAAUCACUGUUGU [762-780](19/19) — — —— 874 human, chimpanzee, GAACUGCUCAUGGACUAGC GCUAGUCCAUGAGCAGUUC[632-650](19/19) — — [752-770](19/19) [614-632] dog (19/19) 875human, chimpanzee, GUGUUUAAGCAGGAGAACU AGUUCUCCUGCUUAAACAC[618-636](19/19) [616-631](16/16) — [738-756](19/19) [600-618] dog(19/19) 876 human, mouse, GAGAAACUGACCCAGAGAA UUCUCUGGGUCAGUUUCUC[447-465](19/19) [445-463](19/19) [326-344](19/19) [567-585](19/19)[429-445] rat, chimpanzee (17/17) 877 human CAAUGUUCAAUUCAGCAAGCUUGCUGAAUUGAACAUUG [2036-2054](19/19) — — — — 878 humanGGCUAUGGAGAUAUGGUUU AAACCAUAUCUCCAUAGCC [1835-1853](19/19) — — — — 879human AUAGGAAAUUAGUUCUGAG CUCAGAACUAAUUUCCUAU [1602-1620](19/19) — — — —880 human GAUAGGAAAUUAGUUCUGA UCAGAACUAAUUUCCUAUC [1601-1619](19/19) — —— — 881 human AUAAACCAGAUUUGCCUAU AUAGGCAAAUCUGGUUUAU [1120-1138](19/19)— — — — 882 human CAACAGUGAUUGAAGGGUC GACCCUUCAAUCACUGUUG[763-781](19/19) — — — — 883 human, chimpanzee UGGUUGCUCAAAGGUCCUUAAGGACCUUUGAGCAACCA [422-440](19/19) [597-607](11/11) [478-488](11/11)[542-560](19/19) [404-422] (18/19) 884 human, chimpanzeeUUGGUUGCUCAAAGGUCCU AGGACCUUUGAGCAACCAA [421-439](19/19)[597-607](11/11) [478-488](11/11) [541-559](19/19) [403-421] (18/19) 885human AUGCUUACAAAAUGGUGAU AUCACCAUUUUGUAAGCAU [2486-2504](19/19) — — — —886 human CUGGAGUUGUCACCACUGA UCAGUGGUGACAACUCCAG [2387-2405](19/19) — —— — 887 human CUCCAAAGGUUCACUGUGU ACACAGUGAACCUUUGGAG [2355-2373](19/19)— — — — 888 human, chimpanzee GAGGUAAUAUUUGAGGAAU AUUCCUCAAAUAUUACCUC[339-357](19/19) — — [459-477](19/19) [321-337] (16/17) 889human, chimpanzee ACGAGGUAAUAUUUGAGGA UCCUCAAAUAUUACCUCGU[337-355](19/19) — — [457-475](19/19) [321-337] (16/17) 890 humanGUUCCUGACUCAAAUUUGA UCAAAUUUGAGUCAGGAAC [1251-1269](19/19) — — — — 891human CUGAUCCUCAGCUCAGGAU AUCCUGAGCUGAGGAUCAG [705-723](19/19)[703-721](18/19) [584-602](18/19) — — 892 human, chimpanzeeAGGUAAUAUUUGAGGAAUC GAUUCCUCAAAUAUUACCU [340-358](19/19) — —[460-478](19/19) [322-337] (15/16) 893 human, chimpanzeeCGAGGUAAUAUUUGAGGAA UUCCUCAAAUAUUACCUCG [338-356](19/19) — —[458-476](19/19) [321-337] (16/17) 894 human AUCAAAACUUCCAAAAGCCGGCUUUUGGAAGUUUUGAU [1222-1240](19/19) — — — [1182-1200] (18/19) 895human UAUCAAAACUUCCAAAAGC GCUUUUGGAAGUUUUGAUA [1221-1239](19/19) — — —[1182-1199] (17/18) 896 human CUGAUUUUCUGGCCUUUGG CCAAAGGCCAGAAAAUCAG[949-967](19/19) — — — — 897 human, chimpanzee, CACUUGUGUUUAAGCAGGAUCCUGCUUAAACACAAGUG [613-631](19/19) [613-629](17/17) — [733-751](19/19)[595-613] dog (19/19) 898 human UAUGCUUACAAAAUGGUGA UCACCAUUUUGUAAGCAUA[2485-2503](19/19) — — — — 899 human GAAUUGAAGUAUCUCUCCUAGGAGAGAUACUUCAAUUC [1737-1755](19/19) — — — — 900 humanUUCCUGACUCAAAUUUGAA UUCAAAUUUGAGUCAGGAA [1252-1270](19/19) — — — — 901human UUCUCUAAGUUUUCAGAGG CCUCUGAAAACUUAGAGAA [1162-1180](19/19) — — — —902 human UGAAUGGGAUGGAAUAGGU ACCUAUUCCAUCCCAUUCA [1024-1042](19/19) — —— — 903 human GAAGUGAUUCAAAAUAGUG CACUAUUUUGAAUCACUUC [969-987](19/19) —— — — 904 human UUGAAGGGUCCUAAAAAGG CCUUUUUAGGACCCUUCAA [772-790](19/19)— — — — 905 human, rat CGACUUGUUAAGAAAAAAC GUUUUUUCUUAACAAGUCG[726-744](19/19) — [605-623](19/19) — [708-724] (16/17) 906human, chimpanzee, UUGUGUUUAAGCAGGAGAA UUCUCCUGCUUAAACACAA[616-634](19/19) [614-631](18/18) — [736-754](19/19) [598-616] dog(19/19) 907 human, chimpanzee, ACACUUGUGUUUAAGCAGG CCUGCUUAAACACAAGUGU[612-630](19/19) [613-628](16/16) — [732-750](19/19) [594-612] dog(19/19) 908 human, chimpanzee UUACACUUGUGUUUAAGCA UGCUUAAACACAAGUGUAA[610-628](19/19) [613-626](14/14) — [730-748](19/19) [594-610] (17/17)909 human, mouse, AGAAACUGACCCAGAGAAU AUUCUCUGGGUCAGUUUCU[448-466](19/19) [446-464](19/19) [327-345](19/19) [568-586](19/19)[430-445] rat, chimpanzee (16/16) 910 human CCAUGGCAGUGUUAUCUCAUGAGAUAACACUGCCAUGG [1701-1719](19/19) — — — — 911 humanAUUGAAGGGUCCUAAAAAG CUUUUUAGGACCCUUCAAU [771-789](19/19) — — — — 912human UGGAACAACAGUGAUUGAA UUCAAUCACUGUUGUUCCA [758-776](19/19) — — —[740-758] (18/19) 913 human, chimpanzee, GCAACUGGCAGUUUGAGCAUGCUCAAACUGCCAGUUGC [210-228](19/19) [208-226](18/19) [89-107](18/19)[330-348](19/19) [192-210] dog (19/19) 914 human, chimpanzeeGGUAAUAUUUGAGGAAUCA UGAUUCCUCAAAUAUUACC [341-359](19/19) — —[461-479](19/19) [327-337] (11/11) 915 human, mouse, rat,CUGAGAAACUGACCCAGAG CUCUGGGUCAGUUUCUCAG [445-463](19/19)[443-461](19/19) [324-342](19/19) [565-583](19/19) [427-445]chimpanzee, dog (19/19) 916 human CUCUGGGCUUUUCUGGGAAUUCCCAGAAAAGCCCAGAG [1721-1739](19/19) — — — — 917 humanUUGGAACAACAGUGAUUGA UCAAUCACUGUUGUUCCAA [757-775](19/19) — — — [739-757](18/19) 918 human UGAUGGCUUAUGGAAGGCU AGCCUUCCAUAAGCCAUCA[2501-2519](19/19) — — — — 919 human GUGAUGGCUUAUGGAAGGCGCCUUCCAUAAGCCAUCAC [2500-2518](19/19) — — — — 920 humanUUGGCUAUGGAGAUAUGGU ACCAUAUCUCCAUAGCCAA [1833-1851](19/19) — — — — 921human CUCUUUUCUUUCUGUGUUU AAACACAGAAAGAAAAGAG [902-920](19/19) — — — —922 human GGAACAACAGUGAUUGAAG CUUCAAUCACUGUUGUUCC [759-777](19/19) — — —[741-759] (18/19) 923 human UCUCUGGGCUUUUCUGGGA UCCCAGAAAAGCCCAGAGA[1720-1738](19/19) — — — — 924 human UCUCUUUUCUUUCUGUGUUAACACAGAAAGAAAAGAGA [901-919](19/19) — — — — 925 humanGUCUCUUUUCUUUCUGUGU ACACAGAAAGAAAAGAGAC [900-918](19/19) — — — —

Note that in the above Table A, the sense strands of siRNAs 1-925 haveSEQ ID NOS: 3-927 respectively, and the antisense strands of siRNAs1-925 have SEQ ID NOS: 928-1852 respectively.

Further note that the coding region of gene RTP801L, as presented inFIG. 1, is between nucleotides 204-785. Therefore, any siRNA within thisregion targets the coding region of RTP801L, and any siRNA outside thisregion targets the non-coding region of RTP801L i.e. the 5′UTR or the 3′UTR. The exact region targeted by each siRNA is given in Table A (column5).

Additionally, all sequences presented in Table A are depicted in thedirection 5′ to 3′.

Example 6

Pharmacology and Drug Delivery

The nucleotide sequences of the present invention are delivered eitherdirectly or with viral or non-viral vectors. When delivered directly thesequences are generally rendered nuclease resistant. Alternatively thesequences can be incorporated into expression cassettes or constructssuch that the sequence is expressed in the cell as discussed hereinbelow. Generally the construct contains the proper regulatory sequenceor promoter to allow the sequence to be expressed in the targeted cell.

The compounds or pharmaceutical compositions of the present inventionare administered and dosed in accordance with good medical practice,taking into account the clinical condition of the individual patient,the disease to be treated, the site and method of administration,scheduling of administration, patient age, sex, body weight and otherfactors known to medical practitioners.

The pharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

The treatment generally has a length proportional to the length of thedisease process and drug effectiveness and the patient species beingtreated. It is noted that humans are treated generally longer than themice or other experimental animals exemplified herein.

The compounds of the present invention are administered by any of theconventional routes of administration. It should be noted that thecompound can be administered as the compound or as pharmaceuticallyacceptable salt and can be administered alone or as an active ingredientin combination with pharmaceutically acceptable carriers, solvents,diluents, excipients, adjuvants and vehicles. The compounds can beadministered orally, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally, andintranasal administration as well as intrathecal and infusiontechniques. Implants of the compounds are also useful. Liquid forms maybe prepared for injection, the term including subcutaneous, transdermal,intravenous, intramuscular, intrathecal, and other parental routes ofadministration. The liquid compositions include aqueous solutions, withand without organic cosolvents, aqueous or oil suspensions, emulsionswith edible oils, as well as similar pharmaceutical vehicles. Inaddition, under certain circumstances the compositions for use in thenovel treatments of the present invention may be formed as aerosols, forintranasal and like administration. The patient being treated is awarm-blooded animal and, in particular, mammals including man. Thepharmaceutically acceptable carriers, solvents, diluents, excipients,adjuvants and vehicles as well as implant carriers generally refer toinert, non-toxic solid or liquid fillers, diluents or encapsulatingmaterial not reacting with the active ingredients of the invention.

When administering the compound of the present invention parenterally,it is generally formulated in a unit dosage injectable form (solution,suspension, emulsion). The pharmaceutical formulations suitable forinjection include sterile aqueous solutions or dispersions and sterilepowders for reconstitution into sterile injectable solutions ordispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, can also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it is desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedhave to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the compounds utilized in the present invention can beadministered parenterally to the patient in the form of slow-releasesubcutaneous implants or targeted delivery systems such as monoclonalantibodies, vectored delivery, iontophoretic, polymer matrices,liposomes, and microspheres. Examples of delivery systems useful in thepresent invention include U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

A pharmacological formulation of the compound utilized in the presentinvention can be administered orally to the patient. Conventionalmethods such as administering the compound in tablets, suspensions,solutions, emulsions, capsules, powders, syrups and the like are usable.Known techniques which deliver it orally or intravenously and retain thebiological activity are preferred. In one embodiment, the compound ofthe present invention can be administered initially by intravenousinjection to bring blood levels to a suitable level. The patient'slevels are then maintained by an oral dosage form, although other formsof administration, dependent upon the patient's condition and asindicated above, can be used.

In general, the active dose of compound for humans is in the range offrom 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of onedose per day or twice or three or more times per day for a period of 1-2weeks or longer, preferably for 24-to 48 hrs or by continuous infusionduring a period of 1-2 weeks or longer.

Administration of Compounds of the Present Invention to the Eye

The compounds of the present invention can be administered to the eyetopically or in the form of an injection, such as an intravitrealinjection, a sub-retinal injection or a bilateral injection. Furtherinformation on administration of the compounds of the present inventioncan be found in Tolentino et al., Retina 24 (2004) 132-138; Reich etal., Molecular vision 9 (2003) 210-216.

Pulmonary Administration of Compounds of the Present Invention

The therapeutic compositions of the present invention are preferablyadministered into the lung by inhalation of an aerosol containing thesecompositions/compounds, or by intranasal or intratracheal instillationof said compositions. Formulating the compositions in liposomes maybenefit absorption. Additionally, the compositions may include a PFCliquid such as perflubron, and the compositions may be formulated as acomplex of the compounds of the invention with polyethylemeimine (PEI).

For further information on pulmonary delivery of pharmaceuticalcompositions see Weiss et al., Human gene therapy 10:2287-2293 (1999);Densmore et al., Molecular therapy 1:180-188 (1999); Gautam et al.,Molecular therapy 3:551-556 (2001); and Shahiwala & Misra, AAPSPharmSciTech 5 (2004). Additionally, respiratory formulations for siRNAare described in U.S. patent application No. 2004/0063654 of Davis etel.

Further, the compounds of the present invention may be administeredtopically where appropriate (such as in the case of diabetic foot ulcersfor example), optionally in a lipid/liposome formulation.

A preferred administration mode is topical delivery of the RTP801Linhibitors onto the round window membrane of the cochlea as disclosedfor example in Tanaka et al. (Hear Res. 2003 March; 177(1-2):21-31).

In the treatment of pressure sores or other wounds, the administrationof the pharmaceutical composition is preferably by topical applicationto the damages area, but the compositions may also be administeredsystemically.

Additional formulations for improved delivery of the compounds of thepresent invention can include non-formulated compounds, compoundscovalently bound to cholesterol, and compounds bound to targetingantibodies (Song et al., Antibody mediated in vivo delivery of smallinterfering RNAs via cell-surface receptors, Nat Biotechnol. 2005 June;23(6):709-17).

Example 7

Model Systems for Pressure Sores or Pressure Ulcers

Pressure sores or pressure ulcers including diabetic ulcers, are areasof damaged skin and tissue that develop when sustained pressure (usuallyfrom a bed or wheelchair) cuts off circulation to vulnerable parts ofthe body, especially the skin on the buttocks, hips and heels. The lackof adequate blood flow leads to ischemic necrosis and ulceration of theaffected tissue. Pressure sores occur most often in patients withdiminished or absent sensation or who are debilitated, emaciated,paralyzed, or long bedridden. Tissues over the sacrum, ischia, greatertrochanters, external malleoli, and heels are especially susceptible;other sites may be involved depending on the patient's situation.

Testing the active inhibitors of the invention (such as siRNA) fortreating pressure sore, ulcers and similar wounds is done in the mousemodel described in Reid R R, Sull A C, Mogford J E, Roy N, Mustoe T A.Cyclical Magnetic Pressure Necrosis: A Novel Murine Model of CutaneousIschemia-Reperfusion Injury. J Surgical Research. 116: 172-180, 2004.

Additionally, a rabbit model is described by Mustoe et al, JCI, 1991;Ahn & Mustoe, Ann Pl Surg, 1991 and is used for testing the siRNAs ofthe invention.

Example 8

Model Systems for Spinal Cord Injury

Spinal cord injury, or myelopathy, is a disturbance of the spinal cordthat results in loss of sensation and/or mobility. The two common typesof spinal cord injury are due to trauma and disease. Traumatic injurycan be due to automobile accidents, falls, gunshot, diving accidentsinter alia, and diseases which can affect the spinal cord include polio,spina bifida, tumors and Friedreich's ataxia.

Testing the active inhibitors of the invention (such as siRNA) fortreating spinal cord injury is done in the rat spinal cord contusionmodel as described by Young, W. in Prog Brain Res. 2002; 137:231-55.Other predictive animal models of spinal cord injury are described inthe following references:

Gruner, J. A. (1992). “A monitored contusion model of spinal cord injuryin the rat.” J Neurotrauma 9(2): 123; Hasegawa, K. and M. Grumet (2003).“Trauma-induced tumorigenesis of cells implanted into the rat spinalcord.” J Neurosurg 98(5): 1065-71; and

Huang, P. P. and W. Young (1994). “The effects of arterial blood gasvalues on lesion volumes in a graded rat spinal cord contusion model.” JNeurotrauma 11(5): 547.

Example 9

Model Systems for Glaucoma

Testing the active inhibitors of the invention (such as siRNA) fortreating or preventing Glaucoma is done in the animal model for exampleas described by Pease et al., J. Glaucoma, 2006, 15(6):512-9 (Manometriccalibration and comparison of TonoLab and TonoPen tonometers in ratswith experimental glaucoma and in normal mice).

Example 10

Model Systems for Ischemia and Reperfusion Injury Following LungTransplantation in Rats

Testing the active inhibitors of the invention (such as siRNA) fortreating or preventing Ischemia and reperfusion injury following lungtransplantation is done in the animal model for example as described byMizobuchi et al., J. Heart Lung Transplant 2004:23:889-93.

Example 11

Model Systems for Acute Lung Injury (ALI)

Intratracheal (i.t) administration of LPS (Lipopolysaccharide), abacterial cell wall component, is an accepted experimental model ofacute lung injury (ALI), as LPS stimulates profound lung recruitment ofinflammatory cells and the subsequent development of systemicinflammation. (See, for example, Fang W F, Cho J H, He Q, Lin M C, Wu CC, Voelkel N F, Douglas I S. “Lipid A Fraction of LPS Induces a DiscreteMAPK Activation in Acute Lung Injury” Am J Physiol Lung Cell MolPhysiol. 2007 May 11; Hagiwara S, Iwasaka H, Noguchi T. “Nafamostatmesilate inhibits the expression of HMGB1 in lipopolysaccharide-inducedacute lung injury” J Anesth. 2007; 21(2):164-70. Epub 2007 May 30.).

Time-dependent changes of RTP801Lgene expression in mice lungs duringthe first 24 hours (time points 0.5; 1; 2; 4; 8 & 24 hours), afterIntratracheal (i.t) administration of LPS was assessed. The assessmentof gene expression was done using qPCR.

The results indicate that the level of the RTP801L transcript isgradually decreased following LPS instillation.

Example 12

Model Systems for Acute Respiratory Distress Syndrome

Testing the active inhibitors of the invention (such as siRNA) fortreating Acute respiratory distress syndrome is done in the animal modelas described by Chen et al. in J Biomed Sci. 2003;10(6 Pt 1):588-92.

Example 13

Model Systems for Hearing Loss Conditions

(i) Distribution of Cy3-PTEN siRNA in the Cochlea Following LocalApplication to the Round Window of the Ear:

A solution of 1 μg/100 μl of Cy3-PTEN siRNA (total of 0.3-0.4 μg) PBSwas applied to the round window of chinchillas. The Cy3-labelled cellswithin the treated cochlea were analyzed 24-48 hours post siRNA roundwindow application after sacrifice of the chinchillas. The pattern oflabeling within the cochlea was similar following 24 h and 48 h andincludes labeling in the basal turn of cochlea, in the middle turn ofcochlea and in the apical turn of cochlea. Application of Cy3-PTEN siRNAonto scala tympani revealed labelling mainly in the basal turn of thecochlea and the middle turn of the cochlea. The Cy3 signal waspersistance to up to 15 days after the application of the Cy3-PTENsiRNA. These results indicate for the first time that local applicationof siRNA molecules within the round window leads to significantpenetration of the siRNA molecules to the basal, middle and apical turnsof the cochlea. The active inhibitors of the invention (such as siRNA)are tested in this model.

(ii) Animal Model of Carboplatin-Induced or Cisplatin-Induced Hair CellDeath in the Cochlea of Chinchilla:

Chinchillas are pre-treated by direct administration of specific siRNAsto 801L (siRNA Nos: 72 or 73 in Table A) in saline to the left ear ofeach animal. Saline is given to the right ear of each animal as placebo.Two days following the administration of the specific siRNA, the animalsare treated with carboplatin (75 mg/kg ip) or cisplatin (intraperitonealinfusion of 13 mg/kg over 30 minutes). After sacrifice of thechinchillas (two weeks post carboplatin treatment) the percentage ofdead cells of inner hair cells (IHC) and outer hair cells (OHC) iscalculated in the left ear (siRNA treated) and in the right ear (salinetreated).The percentage of dead cells is lower in the siRNA treated earthan in the control

(iii) Animal Model of Acoustic-Induced Hair Cell Death in the Cochlea ofChinchilla:

The activity of specific siRNA to 801L (siRNA Nos: 72 or 73 in Table A)in an acoustic trauma model is studied in chinchilla. The animals areexposed to an octave band of noise centered at 4 kHz for 2.5 h at 105dB. The left ear of the noise-exposed chinchillas is pre-treated (48 hbefore the acoustic trauma) with 30 μg of either siRNA in ˜10 μL ofsaline; the right ear is pre-treated with vehicle (saline). The compoundaction potential (CAP) is a convenient and reliable electrophysiologicalmethod for measuring the neural activity transmitted from the cochlea.The CAP is recorded by placing an electrode near the base of the cochleain order to detect the local field potential that is generated when asound stimulus, such as click or tone burst, is abruptly turned on. Thefunctional status of each ear is assessed 2.5 weeks after the acoustictrauma. Specifically, the mean threshold of the compound actionpotential recorded from the round window is determined 2.5 weeks afterthe acoustic trauma in order to determine if the thresholds in thesiRNA-treated ear are lower (better) than the untreated (saline) ear. Inaddition, the amount of inner and outer hair cell loss is determined inthe siRNA-treated and the control ear. It is found that the thresholdsin the siRNA-treated ear are lower than the untreated (saline) ear Also,the amount of hair cell loss is lower in the siRNA-treated ear than inthe control ear.

Example 14

REDD2 siRNA Activity

The following synthetic stabilized siRNA molecules against 801L gene,generated as a stock solution 100 uM in double distilled water, weretested.

SiRNA oligos 1 DDIT4L_216 (table A ID 915) 2 DDIT4L_218 (table A ID 876)3 DDIT4L_15 (table A ID 493) 4 DDIT4L_229 (table A ID 72) 5 DDIT4L_228(table A ID 73)

Thus, the siRNAs tested were blunt-ended 19 mers wherein alternatesugars have a 2′-O-methyl modification, having the sequences presentedin Table A, No.s 915, 876, 493, 72 and 73. The 2-O-methyl modificationis present on nucleotides 1,3,5,7,9,11,13,15,17 and 19 of the antisensestrand and nucleotides 2,4,6,8,10,12,14,16 and 18 of the sense strand.The siRNAs tested were unphosphorylated but the parallel phosphorylatedmolecules are used to achieve essentially identical results. Theinventors of the present application have shown elsewhere (PCT PatentPublication No.s WO 2006/0354 and 2006/023544) that unphosphorylated andthe parallel phosphorylated siRNA modified compounds have similaractivity

The following negative controls were used:

a) Cy3-labeled synthetic stabilized siRNA against human, mouse and ratPTEN gene (PTEN-Cy3). Stock solution 20 mg/ml in double distilled.

b) Synthetic stabilized siRNA against GFP (GFP siRNA). Stock solution 20mg/ml in double distilled.

The cells used in the experiment were 801 wt and Ko mouse embryonicfibroblasts (MEF) cells and 293T embryonic kidney cells.

The transfection reagent used was Lipofectamine 2000 (Invitrogene,Cat#11668-019).

Methods

3×10⁵ and 1×10⁵ 801 wt MEF and 293T cells were seeded per well of the 6wells plate, respectively. 24 h subsequently, cells were transfectedwith:

-   -   a. DDIT4L siRNA molecules at final concentrations per well of        0.5-40 nM    -   b. GFP siRNA molecules at final concentrations per well of        0.5-40 nM    -   c. PTEN-Cy3 siRNA at final concentrations per well of 20-40 nM

Transfection mixture per each well contained 3 μl lipofectamine 2000reagent (in 250 μl serum free medium).

RNA was extracted from cells 72 h following transfection. In the last 8hof incubation, 500 uM H2O2 was added to wt MEF cells.

RNA was prepared from the cells and processed, and qPCR was performedfor the evaluation of REDD2 mRNA levels, using mouse or humanREDD2-specific oligonucleotides and Cyclophylin as a reference gene.

Results and Conclusions

As shown in FIGS. 3-6, the best active siRNA oligos in both mouse andhuman genes were DDIT4L_(—)228 (Table A ID 72) and DDIT4L_(—)229 (TableA ID 73). DDIT4L_(—)228 was slightly more active on the human RTP801Lgene than DDIT4L_(—)229. Both siRNAs 72 and 73 were more active thansiRNAs 915, 876 and 493, as can be seen from FIGS. 3-6.

1. A compound having the structure:

wherein alternating ribonucleotides in the antisense and the sensestrands are modified to result in a 2′-O-methyl modification in thesugar residue of the ribonucleotides; wherein the ribonucleotides at the5′ and 3′ termini of the antisense strand are modified to result in the2′-O-methyl modification; wherein the ribonucleotides at the 5′ and 3′termini of the sense strand are unmodified; wherein at least one of theantisense and the sense strands is phosphorylated at the 3′ termini; andwherein each of the antisense strand and the sense strand is 19-23nucleotides in length.
 2. A composition comprising the compound of claim1 and a pharmaceutically acceptable excipient.
 3. A compound having thestructure:

wherein alternating ribonucleotides in the antisense and the sensestrands are modified to result in a 2′-O-methyl modification in thesugar residue of the ribonucleotides; wherein the ribonucleotides at the5′ and 3′ termini of the antisense strand are modified to result in the2′-O-methyl modification; wherein the ribonucleotides at the 5′ and 3′termini of the sense strand are unmodified; wherein at least one of theantisense and the sense strands is phosphorylated at the 3′ termini; andwherein each of the antisense strand and the sense strand is 19-23nucleotides in length.
 4. A composition comprising the compound of claim3 and a pharmaceutically acceptable excipient.
 5. The compound of claim1, wherein each of the antisense strand and the sense strand is 19nucleotides in length.
 6. The compound of claim 3, wherein each of theantisense strand and the sense strand is 19 nucleotides in length.
 7. Acompound having the structure:

wherein each of the antisense strand and the sense strand is 19-23nucleotides in length.
 8. A compound having the structure:

wherein each of the antisense strand and the sense strand is 19-23nucleotides in length.